A synthetic cytotoxic T cell platform for rapidly prototyping TCR function.

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented a granzyme-activatable sensor of T cell cytotoxicity in a universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24 h exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate knowledge regarding the patterns of T cell receptor recognition, and optimize therapeutic T cell receptors.

Novel Psilocin Prodrugs with Altered Pharmacological Properties as Candidate Therapies for Treatment-Resistant Anxiety Disorders.

The psychedelic prodrug psilocybin has shown therapeutic benefits for the treatment of numerous psychiatric conditions. Despite positive clinical end points targeting depression and anxiety, concerns regarding the duration of the psychedelic experience produced by psilocybin, associated with enduring systemic exposure to the active metabolite psilocin, pose a barrier to its therapeutic application. Our objective was to create a novel prodrug of psilocin with similar therapeutic benefits but a reduced duration of psychedelic effects compared with psilocybin. Here, we report the synthesis and functional screening of 28 new chemical entities. Our strategy was to introduce a diversity of cleavable groups at the 4-hydroxy position of the core indole moiety to modulate metabolic processing. We identified several novel prodrugs of psilocin with altered pharmacokinetic profiles and reduced pharmacological exposure compared with psilocybin. These candidate prodrugs have the potential to maintain the long-term benefits of psilocybin therapy while attenuating the duration of psychedelic effects.

A Synthetic Cytotoxic T cell Platform for Rapidly Prototyping TCR Function.

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented and validated a granzyme-activatable sensor of T cell cytotoxicity in a novel universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered using contemporary gene-editing techniques to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs, with significant responses (p < 0.05 and Cohen's d >1.9) in all cases. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24-hour exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate new knowledge regarding the patterns of T cell receptor recognition, and optimize novel therapeutic T cell receptors for improved cytotoxic potential and reduced cross-reactivity to undesired antigenic targets.

Distinct phosphorylation profiles of tau in brains of patients with different tauopathies.

Tauopathies are neurodegenerative diseases that are characterized by pathological accumulation of tau protein. Tau is hyperphosphorylated in the brain of tauopathy patients, and this phosphorylation is proposed to play a role in disease development. However, it has been unclear whether phosphorylation is different among different tauopathies. Here, we investigated the phosphorylation states of tau in several tauopathies, including corticobasal degeneration, Pick's disease, progressive supranuclear palsy (PSP), argyrophilic grain dementia (AGD) and Alzheimer's disease (AD). Analysis of tau phosphorylation profiles using Phos-tag SDS-PAGE revealed distinct phosphorylation of tau in different tauopathies, whereas similar phosphorylation patterns were found within the same tauopathy. For PSP, we found 2 distinct phosphorylation patterns suggesting that PSP may consist of 2 different related diseases. Immunoblotting with anti-phospho-specific antibodies showed different site-specific phosphorylation in the temporal lobes of patients with different tauopathies. AD brains showed increased phosphorylation at Ser202, Thr231 and Ser235, Pick's disease brains showed increased phospho-Ser202, and AGD brains showed increased phospho-Ser396. The cis conformation of the peptide bond between phospho-Thr231 and Pro232 (cis ptau) was increased in AD and AGD. These results indicate that while tau is differently phosphorylated in tauopathies, a similar pathological mechanism may occur in AGD and AD patients. The present data provide useful information regarding tau pathology and diagnosis of tauopathies.

Genetic Neuropathy Due to Impairments in Mitochondrial Dynamics.

Mitochondria are dynamic organelles capable of fusing, dividing, and moving about the cell. These properties are especially important in neurons, which in addition to high energy demand, have unique morphological properties with long axons. Notably, mitochondrial dysfunction causes a variety of neurological disorders including peripheral neuropathy, which is linked to impaired mitochondrial dynamics. Nonetheless, exactly why peripheral neurons are especially sensitive to impaired mitochondrial dynamics remains somewhat enigmatic. Although the prevailing view is that longer peripheral nerves are more sensitive to the loss of mitochondrial motility, this explanation is insufficient. Here, we review pathogenic variants in proteins mediating mitochondrial fusion, fission and transport that cause peripheral neuropathy. In addition to highlighting other dynamic processes that are impacted in peripheral neuropathies, we focus on impaired mitochondrial quality control as a potential unifying theme for why mitochondrial dysfunction and impairments in mitochondrial dynamics in particular cause peripheral neuropathy.

Characterization of a novel variant in the HR1 domain of MFN2 in a patient with ataxia, optic atrophy and sensorineural hearing loss.

Background: Pathogenic variants in MFN2 cause Charcot-Marie-Tooth disease (CMT) type 2A (CMT2A) and are the leading cause of the axonal subtypes of CMT. CMT2A is characterized by predominantly distal motor weakness and muscle atrophy, with highly variable severity and onset age. Notably, some MFN2 variants can also lead to other phenotypes such as optic atrophy, hearing loss and lipodystrophy. Despite the clear link between MFN2 and CMT2A, our mechanistic understanding of how dysfunction of the MFN2 protein causes human disease pathologies remains incomplete. This lack of understanding is due in part to the multiple cellular roles of MFN2. Though initially characterized for its role in mediating mitochondrial fusion, MFN2 also plays important roles in mediating interactions between mitochondria and other organelles, such as the endoplasmic reticulum and lipid droplets. Additionally, MFN2 is also important for mitochondrial transport, mitochondrial autophagy, and has even been implicated in lipid transfer. Though over 100 pathogenic MFN2 variants have been described to date, only a few have been characterized functionally, and even then, often only for one or two functions. Method: Several MFN2-mediated functions were characterized in fibroblast cells from a patient presenting with cerebellar ataxia, deafness, blindness, and diffuse cerebral and cerebellar atrophy, who harbours a novel homozygous MFN2 variant, D414V, which is found in a region of the HR1 domain of MFN2 where few pathogenic variants occur. Results: We found evidence for impairment of several MFN2-mediated functions. Consistent with reduced mitochondrial fusion, patient fibroblasts exhibited more fragmented mitochondrial networks and had reduced mtDNA copy number. Additionally, patient fibroblasts had reduced oxygen consumption, fewer mitochondrial-ER contacts, and altered lipid droplets that displayed an unusual perinuclear distribution. Conclusion: Overall, this work characterizes D414V as a novel variant in MFN2 and expands the phenotypic presentation of MFN2 variants to include cerebellar ataxia.

Rapid selection and identification of functional CD8+ T cell epitopes from large peptide-coding libraries.

Cytotoxic CD8+ T cells recognize and eliminate infected or malignant cells that present peptide epitopes derived from intracellularly processed antigens on their surface. However, comprehensive profiling of specific major histocompatibility complex (MHC)-bound peptide epitopes that are naturally processed and capable of eliciting a functional T cell response has been challenging. Here, we report a method for deep and unbiased T cell epitope profiling, using in vitro co-culture of CD8+ T cells together with target cells transduced with high-complexity, epitope-encoding minigene libraries. Target cells that are subject to cytotoxic attack from T cells in co-culture are isolated prior to apoptosis by fluorescence-activated cell sorting, and characterized by sequencing the encoded minigenes. We then validate this highly parallelized method using known murine T cell receptor/peptide-MHC pairs and diverse minigene-encoded epitope libraries. Our data thus suggest that this epitope profiling method allows unambiguous and sensitive identification of naturally processed and MHC-presented peptide epitopes.

Tau isoform expression and phosphorylation in marmoset brains.

Tau is a microtubule-associated protein expressed in neuronal axons. Hyperphosphorylated tau is a major component of neurofibrillary tangles, a pathological hallmark of Alzheimer's disease (AD). Hyperphosphorylated tau aggregates are also found in many neurodegenerative diseases, collectively referred to as "tauopathies," and tau mutations are associated with familial frontotemporal lobar degeneration (FTLD). Previous studies have generated transgenic mice with mutant tau as tauopathy models, but nonhuman primates, which are more similar to humans, may be a better model to study tauopathies. For example, the common marmoset is poised as a nonhuman primate model for investigating the etiology of age-related neurodegenerative diseases. However, no biochemical studies of tau have been conducted in marmoset brains. Here, we investigated several important aspects of tau, including expression of different tau isoforms and its phosphorylation status, in the marmoset brain. We found that marmoset tau does not possess the "primate-unique motif" in its N-terminal domain. We also discovered that the tau isoform expression pattern in marmosets is more similar to that of mice than that of humans, with adult marmoset brains expressing only four-repeat tau isoforms as in adult mice but unlike in adult human brains. Of note, tau in brains of marmoset newborns was phosphorylated at several sites associated with AD pathology. However, in adult marmoset brains, much of this phosphorylation was lost, except for Ser-202 and Ser-404 phosphorylation. These results reveal key features of tau expression and phosphorylation in the marmoset brain, a potentially useful nonhuman primate model of neurodegenerative diseases.

Phospho-Tau Bar Code: Analysis of Phosphoisotypes of Tau and Its Application to Tauopathy.

Tau is a microtubule-associated protein which regulates the assembly and stability of microtubules in the axons of neurons. Tau is also a major component of neurofibrillary tangles (NFTs), a pathological hallmark in Alzheimer's disease (AD). A characteristic of AD tau is hyperphosphorylation with more than 40 phosphorylation sites. Aggregates of hyperphosphorylated tau are also found in other neurodegenerative diseases which are collectively called tauopathies. Although a large number of studies have been performed on the phosphorylation of AD tau, it is not known if there is disease-specific phosphorylation among tauopathies. This is due to the lack of a proper method for analyzing tau phosphorylation in vivo. Most previous phosphorylation studies were conducted using a range of phosphorylation site-specific antibodies. These studies describe relative changes of different phosphorylation sites, however, it is hard to estimate total, absolute and collective changes in phosphorylation. To overcome these problems, we have recently applied the Phos-Tag technique to the analysis of tau phosphorylation in vitro and in vivo. This method separates tau into many bands during SDS-PAGE depending on its phosphorylation states, creating a bar code appearance. We propose calling this banding pattern of tau the "phospho-tau bar code." In this review article, we describe what is newly discovered regarding tau phosphorylation through the use of the Phos-Tag. We would like to propose its use for the postmortem diagnosis of tauopathy which is presently done by immunostaining diseased brains with anti-phospho-antibodies. While Phos-tag SDS-PAGE, like other biochemical assays, will lose morphological information, it could provide other types of valuable information such as disease-specific phosphorylation.

Kinase activity of endosomal kinase LMTK1A regulates its cellular localization and interactions with cytoskeletons.

Neurite formation, a fundamental process in neuronal maturation, requires the coordinated regulation of cytoskeletal reorganization and membrane transport. Compared to the understanding of cytoskeletal functions, less is known about the supply of membranes to growing neurites. Lemur kinase 1A (LMTK1A) is an endosomal protein kinase that is highly expressed in neurons. We recently reported that LMTK1A regulates the trafficking of Rab11-positive recycling endosomes in growing axons and dendrites. Here, we used the kinase-negative (kn) mutant to investigate the role of the kinase activity of LMTK1A in its cellular localization and interactions with the cytoskeleton in Neuro2A and PC-12 cells. Kinase activity was required for the localization of LMTK1A in the perinuclear endocytic recycling compartment. Perinuclear accumulation was microtubule dependent, and LMTK1A wild type (wt) localized mainly on microtubules, whereas kn LMTK1A was found in the actin-rich cell periphery. In the neurites of PC-12 cells, LMTK1A showed contrasting distributions depending on the kinase activity, with wt being located in the microtubule-rich shaft and the kn form in the actin-rich tip. Taken together, these results suggest that the kinase activity of LMTK1A regulates the pathway for endosomal vesicles to transfer from microtubules to actin filaments at the tip of growing neurites.

Preferential targeting of p39-activated Cdk5 to Rac1-induced lamellipodia.

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family that plays a role in various neuronal activities including brain development, synaptic regulation, and neurodegeneration. Cdk5 requires the neuronal specific activators, p35 and p39 for subcellular compartmentalization. However, it is not known how active Cdk5 is recruited to F-actin cytoskeleton, which is a Cdk5 target. Here we found p35 and p39 localized to F-actin rich regions of the plasma membrane and investigated the underlying targeting mechanism in vitro by expressing them with Rho family GTPases in Neuro2A cells. Both p35 and p39 accumulated at the cell peripheral lamellipodia and perinuclear regions, where active Rac1 is localized. Interestingly, p35 and p39 displayed different localization patterns as p35 was found more at the perinuclear region and p39 was found more in peripheral lamellipodia. We then confirmed this distinct localization in primary hippocampal neurons. We also determined that the localization of p39 to lamellipodia requires myristoylation and Lys clusters within the N-terminal p10 region. Additionally, we found that p39-Cdk5, but not p35-Cdk5 suppressed lamellipodia formation by reducing Rac1 activity. These results suggest that p39-Cdk5 has a dominant role in Rac1-dependent lamellipodial activity.

Oleanane triterpenes as protein tyrosine phosphatase 1B (PTP1B) inhibitors from Camellia japonica.

Protein tyrosine phosphatase 1B (PTP1B) plays a key role in metabolic signaling, thereby making it an exciting drug target for type 2 diabetes and obesity. Besides, there is substantial evidence that shows its overexpression is involved in breast cancer, which suggests that selective PTP1B inhibition might be effective in breast cancer treatment. As part of our continuous research on PTP1B inhibitors from medicinal plants, four oleanane-type triterpenes were isolated from an EtOAc-soluble extract of fruit peels of Camellia japonica (Theaceae), together with 6 previously known compounds of this class. Their structures were determined on the basis of spectroscopic data analysis (UV, IR, (1)H and (13)CNMR, HMBC, HSQC, NOESY, and MS). All isolates were evaluated for their inhibitory effects on PTP1B, as well as their cytotoxic effects against human breast cancer cell lines MCF7, MCF7/ADR, and MDA-MB-231. Several compounds with OH-3 or/and COOH-28 functionalities showed strong PTP1B inhibitory activity (IC50 values ranging from 3.77±0.11 to 6.40±0.81 µM) as well as significant cytotoxicity (IC50 values ranging from 0.51±0.05 to 13.55±1.44 µM).

T-cell epitope discovery technologies.

Despite tremendous potential utility in clinical medicine and research, the discovery and characterization of T-cell antigens has lagged behind most other areas of health research in joining the high-throughput '-omics' revolution. Partially responsible for this is the complex nature of the interactions between effector T cells and antigen-presenting cells. Further contributing to the challenge is the vastness of both the T-cell repertoire and the large number of potential T-cell epitopes. In this review, we trace the development of various discovery strategies, the technical platforms used to carry them out, and we assess the level of success achieved in the field today.

New prenylated isoflavonoids as protein tyrosine phosphatase 1B (PTP1B) inhibitors from Erythrina addisoniae.

Bioassay-guided fractionation of the EtOAc extract of the root of Erythrina addisoniae (Leguminosae) resulted in the isolation of four new (1-4), along with 2 known prenylated isoflavonoids (5-6). The structures of the isolates were assigned on the basis of spectroscopic data analysis, focusing on interpretation of 1D and 2D NMR, and MS data. All the isolates were evaluated for their inhibitory effects on protein tyrosine phosphatase 1B (PTP1B), as well as their growth inhibition on MCF7, adriamycin-resistant MCF7 (MCF7/ADR), and MDA-MB-231 breast cancer cell lines. Compounds which exhibited PTP1B inhibitory activity (IC(50) values ranging from 4.6 ± 0.3 to 24.2 ± 2.1 µM) showed potential cytotoxic activity (IC(50) values ranging from 3.97 ± 0.17 to 11.4 ± 1.9 µM). Taken together, our data suggest that prenylated isoflavonoids, especially the isoflavone-type skeleton could be considered as new lead compounds against breast cancer via PTP1B inhibition.