Simufilam suppresses overactive mTOR and restores its sensitivity to insulin in Alzheimer's disease patient lymphocytes.

Introduction: Implicated in both aging and Alzheimer's disease (AD), mammalian target of rapamycin (mTOR) is overactive in AD brain and lymphocytes. Stimulated by growth factors such as insulin, mTOR monitors cell health and nutrient needs. A small molecule oral drug candidate for AD, simufilam targets an altered conformation of the scaffolding protein filamin A (FLNA) found in AD brain and lymphocytes that induces aberrant FLNA interactions leading to AD neuropathology. Simufilam restores FLNA's normal shape to disrupt its AD-associated protein interactions. Methods: We measured mTOR and its response to insulin in lymphocytes of AD patients before and after oral simufilam compared to healthy control lymphocytes. Results: mTOR was overactive and its response to insulin reduced in lymphocytes from AD versus healthy control subjects, illustrating another aspect of insulin resistance in AD. After oral simufilam, lymphocytes showed normalized basal mTOR activity and improved insulin-evoked mTOR activation in mTOR complex 1, complex 2, and upstream and downstream signaling components (Akt, p70S6K and phosphorylated Rictor). Suggesting mechanism, we showed that FLNA interacts with the insulin receptor until dissociation by insulin, but this linkage was elevated and its dissociation impaired in AD lymphocytes. Simufilam improved the insulin-mediated dissociation. Additionally, FLNA's interaction with Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), a negative regulator of mTOR, was reduced in AD lymphocytes and improved by simufilam. Discussion: Reducing mTOR's basal overactivity and its resistance to insulin represents another mechanism of simufilam to counteract aging and AD pathology. Simufilam is currently in Phase 3 clinical trials for AD dementia.

Pathway analysis of glutamate-mediated, calcium-related signaling in glioma progression.

Brain tumors, particularly high-grade glioblastomas, are a crucial public health issue due to poor prognosis and an extremely low survival rate. The glioblastoma multiforme (GBM) grows rapidly within its unique microenvironment that is characterized by active neural communications. Therefore, diverse neurotransmitters not only maintain normal brain functions but also influence glioma progression. To fully appreciate the relationship between neurotransmitters and glioma progression, we reviewed potential neurotransmitter contributors in human GBM and the much less aggressive Low-grade glioma (LGG) by combining previously published data from gene-mutation/mRNA sequencing databases together with protein-protein interaction (PPI) network analysis results. The summarized results indicate that glutamatergic and calcium signaling may provide positive feedback to promote glioma formation through 1) metabolic reprogramming and genetic switching to accelerate glioma duplication and progression; 2) upregulation of cytoskeleton proteins and elevation of intracellular Ca2+ levels to increase glutamate release and facilitate formation of synaptic-like connections with surrounding cells in their microenvironment. The upregulated glutamatergic neuronal activities in turn stimulate glioma growth and signaling. Importantly, the enhanced electrical and molecular signals from both neurons and glia propagate out to enable glioma symptoms such as epilepsy and migraine. The elevated intracellular Ca2+ also activates nitric oxide synthase to produce nitric oxide (NO) that can either promote or inhibit tumorigenesis. By analyzing the network effects for complex interaction among neurotransmitters such as glutamate, Ca2+ and NO in brain tumor progression, especially GBM, we identified the glutamatergic signaling as the potential therapeutic targets and suggest manipulation of glutamatergic signaling may be an effective treatment strategy for this aggressive brain cancer.

mGluR5 hypofunction is integral to glutamatergic dysregulation in schizophrenia.

Multiple lines of evidence point to glutamatergic signaling in the postsynaptic density (PSD) as a pathophysiologic mechanism in schizophrenia. Integral to PSD glutamatergic signaling is reciprocal interplay between GluN and mGluR5 signaling. We examined agonist-induced mGluR5 signaling in the postmortem dorsolateral prefrontal cortex (DLPFC) derived from 17 patients and age-matched and sex-matched controls. The patient group showed a striking reduction in mGluR5 signaling, manifested by decreases in Gq/11 coupling and association with PI3K and Homer compared to controls (p < 0.01 for all). This was accompanied by increases in serine and tyrosine phosphorylation of mGluR5, which can decrease mGluR5 activity via desensitization (p < 0.01). In addition, we find altered protein-protein interaction (PPI) of mGluR5 with RGS4, norbin, Preso 1 and tamalin, which can also attenuate mGluR5 activity. We previously reported molecular underpinnings of GluN hypofunction (decreased GluN2 phosphorylation) and here we show those of reduced mGluR5 signaling in schizophrenia. We find that reduced GluN2 phosphorylation can be precipitated by attenuated mGluR5 activity and that increased mGluR5 phosphorylation can result from decreased GluN function, suggesting a reciprocal interplay between the two pathways in schizophrenia. Interestingly, the patient group showed decreased mGluR5-GluN association (p < 0.01), a mechanistic basis for the reciprocal facilitation. In sum, we present the first direct evidence for mGluR5 hypoactivity, propose a reciprocal interplay between GluN and mGluR5 pathways as integral to glutamatergic dysregulation and suggest protein-protein interactions in mGluR5-GluN complexes as potential targets for intervention in schizophrenia.

Insulin and adipokine signaling and their cross-regulation in postmortem human brain.

Aberrant insulin and adipokine signaling has been implicated in cognitive decline associated with both type 2 diabetes mellitus and neurodegenerative diseases. We established methods that reliably measure insulin, adiponectin and leptin signaling, and their crosstalk, in thawed postmortem mid-frontal cortical tissue from cognitively normal older subjects with a short postmortem interval. Insulin-evoked insulin receptor (IR) activation increases activated, tyrosine-phosphorylated IRß on tyrosine residues 960, 1150, and 1151, insulin receptor substrate-1 recruitment to IRß and phosphorylated RAC-α-serine/threonine-protein kinase. Adiponectin augments, but leptin inhibits, insulin signaling. Adiponectin activates adiponectin receptors to induce APPL1 binding to adiponectin receptor 1 and 2 and T-cadherin and downstream adenosine monophosphate-dependent protein kinase phosphorylation. Insulin inhibited adiponectin-induced signaling. In addition, leptin-induced leptin receptor (OB-R) signaling promotes Janus kinase 2 recruitment to OB-R and Janus kinase 2 and downstream signal transducer and activator of transcription 3 phosphorylation. Insulin enhanced leptin signaling. These data demonstrate insulin and adipokine signaling interactions in human brain. Future studies can use these methods to examine insulin, adiponectin, and leptin metabolic dysregulation in aging and disease states, such as type 2 diabetes and Alzheimer's disease-related dementias.

Hyperactivated Insulin Signaling Cascade in Human Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common and malignant glial tumor. Although pro-growth, pro-survival, and pro-metastasis insulin signaling has been proposed to be a prominent driver of GBM progression, the insulin receptor (IR) signaling cascade in GBM has not been fully elucidated. Upon binding of the insulin and insulin-like growth factor-1 (IGF-1), IR is activated by increasing the levels of tyrosine-phosphorylated (pY) IRP on tyrosine 960, 1150, and 1151 residues as well as IRS-1 recruitment to IRß. This leads to activation of the downstream PI3K/AKT/GSK3 or mTORC1/ERK, many of which are implicated in tumorigenesis including breast and liver carcinomas. Here, we directly compare insulin signaling in U87 MG human glioblastoma to primary human astrocytes by assessing the levels of activated IRß, IRS-1 recruitment to IRß, as well as downstream activated mitogenic ERK2 and pro-survival AKT1 under nonstimulated conditions and induced by 1 nM insulin. Our results show insulin receptor and its downstream signaling molecules are robustly hyperactivated. This mechanism renders a reduced insulin-induced response. Our findings provide a mechanism through which GBM develops and grows aggressively even without insulin.

PTI-125 binds and reverses an altered conformation of filamin A to reduce Alzheimer's disease pathogenesis.

We show that amyloid-ß1-42 (Aß42) triggers a conformational change in the scaffolding protein filamin A (FLNA) to induce FLNA associations with α7-nicotinic acetylcholine receptor (α7nAChR) and toll-like receptor 4 (TLR4). These aberrant associations respectively enable Aß42's toxic signaling via α7nAChR to hyperphosphorylate tau protein, and TLR4 activation to release inflammatory cytokines. PTI-125 is a small molecule that preferentially binds altered FLNA and restores its native conformation, restoring receptor and synaptic activities and reducing its α7nAChR/TLR4 associations and downstream pathologies. Two-month oral PTI-125 administration to triple-transgenic (3xTg) Alzheimer's disease (AD) mice before or after apparent neuropathology and to 8-month wildtypes with milder neuropathologies reduced receptor dysfunctions and improved synaptic plasticity, with some improvements in nesting behavior and spatial and working memory in 3xTg AD mice. PTI-125 also reduced tau hyperphosphorylation, aggregated Aß42 deposition, neurofibrillary tangles, and neuroinflammation. Efficacy in postmortem AD and Aß42-treated age-matched control hippocampal slices was concentration-dependent starting at 1 picomolar (pM) concentration. PTI-125 is the first therapeutic candidate to preferentially bind an altered protein conformation and reverse this proteopathy.

P53 regulates the migration of mesenchymal stromal cells in response to the tumor microenvironment through both CXCL12-dependent and -independent mechanisms.

Mesenchymal stromal cells (MSCs) are multipotent fibroblast-like cells located in the bone marrow that localize to areas of tissue damage including wounds and solid tumors. Within the tumor microenvironment, MSCs adopt the phenotype of carcinoma-associated fibroblasts (CAFs) and stimulate tumor growth. Production of the chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF-1), by MSCs is required for their in vitro migration in response to tumor cells and has also been implicated in stimulation of tumor growth. The tumor suppressor p53 regulates cellular migration, CXCL12 production and the promotion of tumor growth by carcinoma-associated fibroblasts (CAFs). We investigated the role of p53 in MSC migration to tumors. P53 inhibits the migration of MSCs in response to tumor cells in conjunction with a decrease in CXCL12 transcription. Conversely, decreased p53 activity leads to enhanced MSC migration. Interestingly, increased p53 activity inhibits MSC migration even in the context of high concentrations of exogenous CXCL12. These data show that stromal p53 status impacts the recruitment of MSCs to solid tumors through both regulation of CXCL12 production as well as other mechanisms. Stromal p53 may influence other important aspects of tumor biology such as tumor growth and metastasis through mechanisms distinct from CXCL12.

Antitumor and modeling studies of a penetratin-peptide that targets E2F-1 in small cell lung cancer.

E2F-1, a key transcription factor necessary for cell growth, DNA repair, and differentiation, is an attractive target for development of anticancer drugs in tumors that are E2F "oncogene addicted". We identified a peptide isolated from phage clones that bound tightly to the E2F-1 promoter consensus sequence. The peptide was coupled to penetratin to enhance cellular uptake. Modeling of the penetratin-peptide (PEP) binding to the DNA E2F-1 promoter demonstrated favorable interactions that also involved the participation of most of the penetratin sequence. The penetratin-peptide (PEP) demonstrated potent in vitro cytotoxic effects against a range of cancer cell lines, particularly against Burkitt lymphoma cells and small cell lung cancer (SCLC) cells. Further studies in the H-69 SCLC cell line showed that the PEP inhibited transcription of E2F-1 and also several important E2F-regulated enzymes involved in DNA synthesis, namely, thymidylate synthase, thymidine kinase, and ribonucleotide reductase. As the PEP was found to be relatively unstable in serum, it was encapsulated in PEGylated liposomes for in vivo studies. Treatment of mice bearing the human small cell lung carcinoma H-69 with the PEP encapsulated in PEGylated liposomes (PL-PEP) caused tumor regression without significant toxicity. The liposome encapsulated PEP has promise as an antitumor agent, alone or in combination with inhibitors of DNA synthesis.

Isoflurane increases neuronal cell death vulnerability by downregulating miR-214.

Since accumulating evidence suggests the application of anesthetics may increase the risk of Alzheimer's disease (AD), we investigated the cytotoxicity of inhaled general anesthesia in neurons and its underlying mechanism. Using primary cultured rat hippocampal neurons as the study model, here we show that isoflurane increases vulnerability to intracellular or extracellular amyloid ß with or without serum deprivation. This isoflurane-induced effect is mediated by the downregulation of miR-214 level that lead to an elevated expression of Bax, a prominent target for miR-214. We conclude that isoflurane increases cell death in the presence of amyloid ß by increasing Bax level through downregulating miR-214. Our data provide a new insight for inhaled anesthetics toxicity and indicate a possible mechanistic link between anesthetic application and neurodegenration in AD.

Enhanced degradation of dihydrofolate reductase through inhibition of NAD kinase by nicotinamide analogs.

Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin's lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment.