Protein interacting with C kinase (PICK1) is a scaffolding protein that is present in dendritic spines and interacts with a wide array of proteins through its PDZ domain. The best understood function ...of PICK1 is regulation of trafficking of AMPA receptors at neuronal synapses via its specific interaction with the AMPA GluA2 subunit. Disrupting the PICK1-GluA2 interaction has been shown to alter synaptic plasticity, a molecular mechanism of learning and memory. Lack of potent, selective inhibitors of the PICK1 PDZ domain has hindered efforts at exploring the PICK1-GluA2 interaction as a therapeutic target for neurological diseases. Here, we report the discovery of PICK1 small molecule inhibitors using a structure-based drug design strategy. The inhibitors stabilized surface GluA2, reduced Aβ-induced rise in intracellular calcium concentrations in cultured neurons, and blocked long term depression in brain slices. These findings demonstrate that it is possible to identify potent, selective PICK1-GluA2 inhibitors which may prove useful for treatment of neurodegenerative disorders.
Inhibition of heat shock protein 90 (Hsp90) results in the degradation of oncoproteins that drive malignant progression, inducing
cell death, making Hsp90 a target of substantial interest for cancer ...therapy. BIIB021 is a novel, fully synthetic inhibitor
of Hsp90 that binds competitively with geldanamycin in the ATP-binding pocket of Hsp90. In tumor cells, BIIB021 induced the
degradation of Hsp90 client proteins including HER-2, AKT, and Raf-1 and up-regulated expression of the heat shock proteins
Hsp70 and Hsp27. BIIB021 treatment resulted in growth inhibition and cell death in cell lines from a variety of tumor types
at nanomolar concentrations. Oral administration of BIIB021 led to the degradation of Hsp90 client proteins measured in tumor
tissue and resulted in the inhibition of tumor growth in several human tumor xenograft models. Studies to investigate the
antitumor effects of BIIB021 showed activity on both daily and intermittent dosing schedules, providing dose schedule flexibility
for clinical studies. Assays measuring the HER-2 protein in tumor tissue and the HER-2 extracellular domain in plasma were
used to show interdiction of the Hsp90 pathway and utility as potential biomarkers in clinical trials for BIIB021. Together,
these data show that BIIB021 is a promising new oral inhibitor of Hsp90 with antitumor activity in preclinical models.Mol
Cancer Ther 2009;8(4):921–9
The discrete localization of ion channels is a critical determinant of neuronal excitability. We show here that the dendritic K
+ channels Kv2.1 and Kv2.2 were differentially targeted in cultured ...hippocampal neurons. Kv2.1 was found in high-density clusters on the soma and proximal dendrites, while Kv2.2 was uniformly distributed throughout the soma and dendrites. Chimeras revealed a proximal restriction and clustering domain on the cytoplasmic tail of Kv2.1. Truncations and internal deletions revealed a 26–amino acid targeting signal within which four residues were critical for localization. This signal is not related to other known sequences for neuronal and epithelial membrane protein targeting and represents a novel cytoplasmic signal responsible for proximal restriction and clustering.
There are currently no preventive or disease-modifying therapies for Parkinson's Disease (PD). Failures in clinical trials necessitate a re-evaluation of existing pre-clinical models in order to ...adopt systems that better recapitulate underlying disease mechanisms and better predict clinical outcomes. In recent years, models utilizing patient-derived induced pluripotent stem cells (iPSC) have emerged as attractive models to recapitulate disease-relevant neuropathology in vitro without exogenous overexpression of disease-related pathologic proteins. Here, we utilized iPSC derived from patients with early-onset PD and dementia phenotypes that harbored either a point mutation (A53T) or multiplication at the α-synuclein/SNCA gene locus. We generated a three-dimensional (3D) cortical neurosphere culture model to better mimic the tissue microenvironment of the brain. We extensively characterized the differentiation process using quantitative PCR, Western immunoblotting and immunofluorescence staining. Differentiated and aged neurospheres revealed alterations in fatty acid profiles and elevated total and pathogenic phospho-α-synuclein levels in both A53T and the triplication lines compared to their isogenic control lines. Furthermore, treatment of the neurospheres with a small molecule inhibitor of stearoyl CoA desaturase (SCD) attenuated the protein accumulation and aberrant fatty acid profile phenotypes. Our findings suggest that the 3D cortical neurosphere model is a useful tool to interrogate targets for PD and amenable to test small molecule therapeutics.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To test the hypothesis that dimethyl fumarate (DMF, Tecfidera) elicits different biological changes from DMF combined with monoethyl fumarate (MEF) (Fumaderm, a psoriasis therapy), we investigated ...DMF and MEF in rodents and cynomolgus monkeys. Possible translatability of findings was explored with lymphocyte counts from a retrospective cohort of patients with MS.
In rodents, we evaluated pharmacokinetic and pharmacodynamic effects induced by DMF and MEF monotherapies or in combination (DMF/MEF). Clinical implications were investigated in a retrospective, observational analysis of patients with MS treated with DMF/MEF (n = 36).
In rodents and cynomolgus monkeys, monomethyl fumarate (MMF, the primary metabolite of DMF) exhibited higher brain penetration, whereas MEF was preferentially partitioned into the kidney. In mice, transcriptional profiling for DMF and MEF alone identified both common and distinct pharmacodynamic responses, with almost no overlap between DMF- and MEF-induced differentially expressed gene profiles in immune tissues. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated oxidative stress response pathway was exclusively regulated by DMF, whereas apoptosis pathways were activated by MEF. DMF/MEF treatment demonstrated that DMF and MEF functionally interact to modify DMF- and MEF-specific responses in unpredictable ways. In patients with MS, DMF/MEF treatment led to early and pronounced suppression of lymphocytes, predominantly CD8
T cells. In a multivariate regression analysis, the absolute lymphocyte count (ALC) was associated with age at therapy start, baseline ALC, and DMF/MEF dosage but not with previous immunosuppressive medication and sex. Furthermore, the ALC increased in a small cohort of patients with MS (n = 6/7) after switching from DMF/MEF to DMF monotherapy.
Fumaric acid esters exhibit different biodistribution and may elicit different biological responses; furthermore, pharmacodynamic effects of combinations differ unpredictably from monotherapy. The strong potential to induce lymphopenia in patients with MS may be a result of activation of apoptosis pathways by MEF compared with DMF.
The development of phenotypic assays with appropriate analyses is an important step in the drug discovery process. Assays using induced pluripotent stem cell (iPSC)-derived human neurons are emerging ...as powerful tools for drug discovery in neurological disease. We have previously shown that longitudinal single cell tracking enabled the quantification of survival and death of neurons after overexpression of α-synuclein with a familial Parkinson's disease mutation (A53T). The reliance of this method on manual counting, however, rendered the process labor intensive, time consuming and error prone. To overcome these hurdles, we have developed automated detection algorithms for neurons using the BioStation CT live imaging system and CL-Quant software. In the current study, we use these algorithms to successfully measure the risk of neuronal death caused by overexpression of α-synuclein (A53T) with similar accuracy and improved consistency as compared to manual counting. This novel method also provides additional key readouts of neuronal fitness including total neurite length and the number of neurite nodes projecting from the cell body. Finally, the algorithm reveals the neuroprotective effects of brain-derived neurotrophic factor (BDNF) treatment in neurons overexpressing α-synuclein (A53T). These data show that an automated algorithm improves the consistency and considerably shortens the analysis time of assessing neuronal health, making this method advantageous for small molecule screening for inhibitors of synucleinopathy and other neurodegenerative diseases.
Plasminogen activators are used in thrombolytic stroke therapy. However, it is increasingly recognized that they have other actions besides fibrinolysis. In this study, we assess potential ...pro-inflammatory effects of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in rat cortical astrocytes. Both uPA and tPA induced rapid dose-dependent upregulation in MMP-2 and MMP-9, as demonstrated by zymography of conditioned media. In addition, a multiplex ELISA array demonstrated that patterned responses in chemokines and cytokines were also evoked. Exposure to tPA induced elevations in secreted MIP-2, MCP-1 and GRO/KC. Exposure to uPA induced elevations in secreted IFN-γ, TNF-α, GMCSF, MIP-1α, MIP-2, MIP-3α, MCP-1, RANTES and fractalkine. These data suggest that plasminogen activators may trigger selected pro-inflammatory responses at the neurovascular interface. Whether these effects influence thrombolytic stroke therapy warrants further investigation.
Reducing the production of larger aggregation-prone amyloid β-peptides (Aβ) remains an untested therapeutic approach for reducing the appearance and growth of Aβ plaques in the brain, which are a ...hallmark pathological feature of Alzheimer's disease. γ-Secretase modulators (GSMs) are therapeutics that impact γ-secretase-dependent cleavage of amyloid precursor protein to promote the production of shorter Aβ peptides that are less prone to aggregation and plaque deposition. This is accomplished without inhibiting overall γ-secretase function and cleavage of other substrates, which is believed to be a source of deleterious side effects. Here, we report the pharmacokinetic and pharmacodynamic properties of BIIB042, a novel bioavailable and brain-penetrant GSM. In cell-based assays, BIIB042 reduced the levels of Aβ42, increased the levels of Aβ38 and had little effect on the levels of Aβ40, the most abundant Aβ species. Similar pharmacodynamic properties were confirmed in the central nervous system and in plasma of mice and rats, and also in plasma of cynomolgus monkeys after a single oral dose of BIIB042. BIIB042 reduced Aβ42 levels and Aβ plaque burden in Tg2576 mice, which overexpress human amyloid precursor protein and serve as a model system for Alzheimer's disease. BIIB042 did not inhibit cleavage of other γ-secretase substrates in cell-based and in vivo signaling and cleavage assays. The pharmacodynamic effects of lowering Aβ42 in the central nervous system coupled with demonstrated efficacy in reducing plaque pathology suggests modulation of γ-secretase, with molecules like BIIB042, is a compelling therapeutic approach for the treatment of Alzheimer's disease.
•Longer amyloid peptides (Aβ42) are thought to by pathogenic in Alzheimer's disease.•γ-Secretase modulators selectively reduce pathogenic Aβ isoforms like Aβ42.•BIIB042 is a potent, brain penetrant γ-secretase modulator.•BIIB042 reduced Aβ42 levels in cell models, rodents and cynomolgus monkeys.•BIIB042 reduced plaque pathology in a mouse transgenic model of Alzheimer's disease.
The family of calcium binding proteins called KChIPs associates with Kv4 family K
+ channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore ...the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7–11 and 71–90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71–90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71–90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.