Jacob-induced transcriptional inactivation of CREB promotes Aß-induced synapse loss in Alzheimer's disease.

Synaptic dysfunction caused by soluble ß-amyloid peptide (Aß) is a hallmark of early-stage Alzheimer's disease (AD), and is tightly linked to cognitive decline. By yet unknown mechanisms, Aß suppresses the transcriptional activity of cAMP-responsive element-binding protein (CREB), a master regulator of cell survival and plasticity-related gene expression. Here, we report that Aß elicits nucleocytoplasmic trafficking of Jacob, a protein that connects a NMDA-receptor-derived signalosome to CREB, in AD patient brains and mouse hippocampal neurons. Aß-regulated trafficking of Jacob induces transcriptional inactivation of CREB leading to impairment and loss of synapses in mouse models of AD. The small chemical compound Nitarsone selectively hinders the assembly of a Jacob/LIM-only 4 (LMO4)/ Protein phosphatase 1 (PP1) signalosome and thereby restores CREB transcriptional activity. Nitarsone prevents impairment of synaptic plasticity as well as cognitive decline in mouse models of AD. Collectively, the data suggest targeting Jacob protein-induced CREB shutoff as a therapeutic avenue against early synaptic dysfunction in AD.

Dipeptidyl peptidase 4 deficiency improves survival after focal cerebral ischemia in mice and ameliorates microglia activation and specific inflammatory markers.

Dipeptidyl peptidase 4 (DPP4; CD26) is involved in the regulation of various metabolic, immunological, and neurobiological processes in healthy individuals. Observations based on epidemiological data indicate that DPP4 inhibition by gliptins, typically used in patients with diabetes, may reduce the risk for cerebral ischemia and may also improve related outcomes. However, as DPP4 inhibitor application is neither complete nor specific for suppression of DPP4 enzymatic activity and DPP4 has non-enzymatic functions as well, the variety of consequences is a matter of debate. Therefore, we here used DPP4 knock-out (KO) mice to analyze the specific contribution of DPP4 to cellular, immunological, and functional consequences of experimental focal cerebral ischemia. We observed a significantly higher survival rate of DPP4 KO mice after ischemia, which was accompanied by a lower abundance of the pro-inflammatory chemokine CCL2 and reduced activation of Iba1-positive microglia cells in brain tissue of DPP4 KO mice. In addition, after ischemia for 24 h to 72 h, decreased concentrations of CCL5 and CCL12 in plasma and of CCL17 in brain tissue of DPP4 KO mice were observed when compared to wild type mice. Other aspects analyzed, such as the functional Menzies score, astrocyte activation and chemokine levels in plasma and brain tissue were affected by ischemia but appeared to be unaffected by the DPP4 KO genotype. Taken together, experimental ablation of DPP4 functions in mice improves survival and ameliorates aspects of cellular and molecular inflammation after focal cerebral ischemia.

Tricellulin, α-Catenin and Microfibrillar-Associated Protein 5 Exhibit Concomitantly Altered Immunosignals along with Vascular, Extracellular and Cytoskeletal Elements after Experimental Focal Cerebral Ischemia.

Along with initiatives to understand the pathophysiology of stroke in detail and to identify neuroprotective targets, cell-stabilizing elements have gained increasing attention. Although cell culture experiments have indicated that tricellulin, α-catenin and microfibrillar-associated protein 5 (MFAP5) contribute to cellular integrity, these elements have not yet been investigated in the ischemic brain. Applying immunofluorescence labeling, this study explored tricellulin, MFAP5 and α-catenin in non-ischemic and ischemic brain areas of mice (24, 4 h of ischemia) and rats (4 h of ischemia), along with collagen IV and fibronectin as vascular and extracellular matrix constituents and microtubule-associated protein 2 (MAP2) and neurofilament light chain (NF-L) as cytoskeletal elements. Immunosignals of tricellulin and notably MFAP5 partially appeared in a fiber-like pattern, and α-catenin appeared more in a dotted pattern. Regional associations with vascular and extracellular constituents were found for tricellulin and α-catenin, particularly in ischemic areas. Due to ischemia, signals of tricellulin, MFAP5 and α-catenin decreased concomitantly with MAP2 and NF-L, whereby MFAP5 provided the most sensitive reaction. For the first time, this study demonstrated ischemia-related alterations in tricellulin, MFAP5 and α-catenin along with the vasculature, extracellular matrix and cytoskeleton. Confirmatory studies are needed, also exploring their role in cellular integrity and the potential for neuroprotective approaches in stroke.

Immunogenicity of the Envelope Surface Unit of Human Endogenous Retrovirus K18 in Mice.

The triggers for the development of multiple sclerosis (MS) have not been fully understood to date. One hypothesis proposes a viral etiology. Interestingly, viral proteins from human endogenous retroviruses (HERVs) may play a role in the pathogenesis of MS. Allelic variants of the HERV-K18 env gene represent a genetic risk factor for MS, and the envelope protein is considered to be an Epstein-Barr virus-trans-activated superantigen. To further specify a possible role for HERV-K18 in MS, the present study examined the immunogenicity of the purified surface unit (SU). HERV-K18(SU) induced envelope-specific plasma IgG in immunized mice and triggered proliferation of T cells isolated from these mice. It did not trigger phenotypic changes in a mouse model of experimental autoimmune encephalomyelitis. Further studies are needed to investigate the underlying mechanisms of HERV-K18 interaction with immune system regulators in more detail.

A novel compound heterozygous leptin receptor mutation causes more severe obesity than in Leprdb/db mice.

The leptin receptor (Lepr) pathway is important for food intake regulation, energy expenditure, and body weight. Mutations in leptin and the Lepr have been shown to cause early-onset severe obesity in mice and humans. In studies with C57BL/6NCrl mice, we found a mouse with extreme obesity. To identify a putative spontaneous new form of monogenic obesity, we performed backcross studies with this mouse followed by a quantitative trait locus (QTL) analysis and sequencing of the selected chromosomal QTL region. We thereby identified a novel Lepr mutation (C57BL/6N-LeprL536Hfs*6-1NKB), which is located at chromosome 4, exon 11 within the CRH2-leptin-binding site. Compared with C57BL/6N mice, LeprL536Hfs*6 develop early onset obesity and their body weight exceeds that of Leprdb/db mice at an age of 30 weeks. Similar to Leprdb/db mice, the LeprL536Hfs*6 model is characterized by hyperphagia, obesity, lower energy expenditure and activity, hyperglycemia, and hyperinsulinemia compared with C57BL/6N mice. Crossing Leprdb/wt with LeprL536Hfs*6/wt mice results in compound heterozygous LeprL536Hfs*6/db mice, which develop even higher body weight and fat mass than both homozygous Leprdb/db and LeprL536Hfs*6 mice. Compound heterozygous Lepr deficiency affecting functionally different regions of the Lepr causes more severe obesity than the parental homozygous mutations.

Construction of a 3D brain extracellular matrix model to study the interaction between microglia and T cells in co-culture.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

A glutaminyl cyclase-catalyzed α-synuclein modification identified in human synucleinopathies.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79-α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may-in analogy to pGlu-Aß peptides in Alzheimer's disease-act as a seed for pathogenic protein aggregation.

Proteolytic α-Synuclein Cleavage in Health and Disease.

In Parkinson's disease, aggregates of α-synuclein within Lewy bodies and Lewy neurites represent neuropathological hallmarks. However, the cellular and molecular mechanisms triggering oligomeric and fibrillary α-synuclein aggregation are not fully understood. Recent evidence indicates that oxidative stress induced by metal ions and post-translational modifications such as phosphorylation, ubiquitination, nitration, glycation, and SUMOylation affect α-synuclein conformation along with its aggregation propensity and neurotoxic profiles. In addition, proteolytic cleavage of α-synuclein by specific proteases results in the formation of a broad spectrum of fragments with consecutively altered and not fully understood physiological and/or pathological properties. In the present review, we summarize the current knowledge on proteolytical α-synuclein cleavage by neurosin, calpain-1, cathepsin D, and matrix metalloproteinase-3 in health and disease. We also shed light on the contribution of the same enzymes to proteolytical processing of pathogenic proteins in Alzheimer's disease and report potential cross-disease mechanisms of pathogenic protein aggregation.

Formation of HERV-K and HERV-Fc1 Envelope Family Members is Suppressed on Transcriptional and Translational Level.

The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.

Amyloid-Beta Peptides Trigger Aggregation of Alpha-Synuclein In Vitro.

Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aß(1-42) and pGlu-Aß(3-42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aß(1-42) and pGlu-Aß(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aß in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aß and pGlu-Aß, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aß species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.