Casein kinase 1 inhibitor avoids TDP-43 pathology propagation in a patient-derived cellular model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease without a cure to reverse its progression. Its main hallmark is the nuclear protein TDP-43, which undergoes different post-translational modifications leading to a loss of function in the nucleus and an increase in toxicity in the cytoplasm. Previous reports have indicated that pathogenic TDP-43 exhibits prion-like propagation in various contexts. With the aim of advancing therapeutics focused on preventing the propagation of TDP-43 pathology, we studied the potential role of pathogenic TDP-43 in lymphoblasts from sporadic ALS patients. We used lymphoblastoid cell lines from sporadic ALS patients as a source of pathogenic forms of TDP-43, and healthy human cells (lymphoblasts, myoblasts, neuroblastoma SH-SY5Y, or osteosarcoma U2OS) as recipient cells to investigate the seeding and spread of TDP-43 proteinopathy. Furthermore, we evaluated the potential of targeting TDP-43 phosphorylation with a CK-1 inhibitor to prevent the propagation of the pathology. The results presented herein indicate that pathogenic forms of TDP-43 are secreted into the extracellular medium of sporadic ALS lymphoblasts and could be transported by extracellular vesicles, spreading TDP-43 pathology to healthy cells. Moreover, tunneling nanotubes have also been discovered in pathological cells and may be involved in the transport of TDP-43. Interestingly, targeting TDP-43 phosphorylation with an in-house designed CK-1 inhibitor (IGS2.7) was sufficient to halt TDP-43 pathology transmission, in addition to its known effects on restoring the homeostasis of TDP-43 protein in patients-derived cells.

Functional Characterization of a Familial ALS-Associated Missense TBK1 (p-Arg573Gly) Mutation in Patient-Derived Lymphoblasts.

The goal of this work was to elucidate the pathogenic mechanism of an ALS-associated missense mutation, p.Arg573Gly (R573G), in the TBK1 gene. In particular, we seek to analyze the influence of this variant on the cellular levels and the function of TBK1 in immortalized cells from an ALS patient. The patient (Code# E7) belonged to a Spanish family with autosomal dominant disease manifesting in the sixth decade as either dementia or ALS. Four control individuals without signs of neurological disease were also included in this study. Our results indicate that the R375G TBK1 mutation did not affect the levels of mRNA nor the total TBK1 content; however, we observed a significant decrease in the levels of TBK1 phosphorylation, which is essential for TBK1 activity, as well as a significant reduction in the phosphorylation of p62 and RIPK1, known substrates for TBK1. Lymphoblasts from the R573G TBK1 mutation carrier patient display pathological TDP-43 homeostasis, showing elevated levels of phosphorylated TDP-43 and accumulation of the protein in the cytosolic compartment. In addition, the functional decrease in TBK1 activity observed in the E7 patient did not alter the autophagy flux, but it seems to be enough to increase ROS levels as well as the expression of pro-inflammatory cytokine IL-6.

Multitarget drugs as potential therapeutic agents for alzheimer's disease. A new family of 5-substituted indazole derivatives as cholinergic and BACE1 inhibitors.

Multitarget drugs are a promising therapeutic approach against Alzheimer's disease. In this work, a new family of 5-substituted indazole derivatives with a multitarget profile including cholinesterase and BACE1 inhibition is described. Thus, the synthesis and evaluation of a new class of 5-substituted indazoles has been performed. Pharmacological evaluation includes in vitro inhibitory assays on AChE/BuChE and BACE1 enzymes. Also, the corresponding competition studies on BuChE were carried out. Additionally, antioxidant properties have been calculated from ORAC assays. Furthermore, studies of anti-inflammatory properties on Raw 264.7 cells and neuroprotective effects in human neuroblastoma SH-SY5Y cells have been performed. The results of pharmacological tests have shown that some of these 5-substituted indazole derivatives 1-4 and 6 behave as AChE/BuChE and BACE1 inhibitors, simultaneously. In addition, some indazole derivatives showed anti-inflammatory (3, 6) and neuroprotective (1-4 and 6) effects against Aß-induced cell death in human neuroblastoma SH-SY5Y cells with antioxidant properties.

Impact of Multiple Sclerosis Risk Polymorphism rs7665090 on MANBA Activity, Lysosomal Endocytosis, and Lymphocyte Activation.

Deficiencies in Mannosidase ß (MANBA) are associated with neurological abnormalities and recurrent infections. The single nucleotide polymorphism located in the 3'UTR of MANBA, rs7665090, was found to be associated with multiple sclerosis (MS) susceptibility. We aimed to study the functional impact of this polymorphism in lymphocytes isolated from MS patients and healthy controls. A total of 152 MS patients and 112 controls were genotyped for rs7665090. MANBA mRNA expression was quantified through qPCR and MANBA enzymatic activity was analyzed. Upon phytohemagglutinin stimulation, immune activation was evaluated by flow cytometry detection of CD69, endocytic function, and metabolic rates with Seahorse XFp Analyzer, and results were stratified by variation in rs7665090. A significantly reduced gene expression (p < 0.0001) and enzymatic activity (p = 0.018) of MANBA were found in lymphocytes of MS patients compared to those of controls. The rs7665090*GG genotype led to a significant ß-mannosidase enzymatic deficiency correlated with lysosomal dysfunction, as well as decreased metabolic activation in lymphocytes of MS patients compared to those of rs7665090*GG controls. In contrast, lymphocytes of MS patients and controls carrying the homozygous AA genotype behaved similarly. Our work provides new evidence highlighting the impact of the MS-risk variant, rs7665090, and the role of MANBA in the immunopathology of MS.

Therapeutic potential of novel Cell Division Cycle Kinase 7 inhibitors on TDP-43-related pathogenesis such as Frontotemporal Lobar Degeneration (FTLD) and amyotrophic lateral sclerosis (ALS).

TDP-43 has been identified as the major component of protein aggregates found in affected neurons in FTLD-TDP and amyotrophic lateral sclerosis (ALS) patients. TDP-43 is hyperphosphorylated, ubiquitinated, and cleaved in the C-terminus. CDC-7 was reported to phosphorylate TDP-43. There are no effective treatments for either FTLD-TDP or ALS, being a pressing need for the search of new therapies. We hypothesized that modulating CDC-7 activity with small molecules that are able to interfere with TDP-43 phosphorylation could be a good therapeutic strategy for these diseases. Here, we have studied the effects of novel brain penetrant, thiopurine-based, CDC-7 inhibitors in TDP-43 homeostasis in immortalized lymphocytes from FTLD-TDP patients, carriers of a loss-of-function GRN mutation, as well as in cells derived from sporadic ALS patients. We found that selective CDC-7 inhibitors, ERP1.14a and ERP1.28a, are able to decrease the enhanced TDP-43 phosphorylation in cells derived from FTLD-TDP and ALS patients and to prevent cytosolic accumulation of TDP-43. Moreover, treatment of FTLD-TDP lymphoblasts with CDC-7 inhibitors leads to recovering the nuclear function of TDP-43-inducing CDK6 repression. We suggest that CDC-7 inhibitors, mainly the heterocyclic compounds here shown, may be considered as promising drug candidates for the ALS/FTD spectrum.

Tideglusib, a Non-ATP Competitive Inhibitor of GSK-3ß as a Drug Candidate for the Treatment of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is the most common degenerative motor neuron disease in adults. About 97% of ALS patients present TDP-43 aggregates with post-translational modifications, such as hyperphosphorylation, in the cytoplasm of affected cells. GSK-3ß is one of the protein kinases involved in TDP-43 phosphorylation. Up-regulation of its expression and activity is reported on spinal cord and cortex tissues of ALS patients. Here, we propose the repurposing of Tideglusib, an in-house non-ATP competitive GSK-3ß inhibitor that is currently in clinical trials for autism and myotonic dystrophy, as a promising therapeutic strategy for ALS. With this aim we have evaluated the efficacy of Tideglusib in different experimental ALS models both in vitro and in vivo. Moreover, we observed that GSK-3ß activity is increased in lymphoblasts from sporadic ALS patients, with a simultaneous increase in TDP-43 phosphorylation and cytosolic TDP-43 accumulation. Treatment with Tideglusib decreased not only phospho-TDP-43 levels but also recovered its nuclear localization in ALS lymphoblasts and in a human TDP-43 neuroblastoma model. Additionally, we found that chronic oral treatment with Tideglusib is able to reduce the increased TDP-43 phosphorylation in the spinal cord of Prp-hTDP-43A315T mouse model. Therefore, we consider Tideglusib as a promising drug candidate for ALS, being proposed to start a clinical trial phase II by the end of the year.

Molecular Alterations in Sporadic and SOD1-ALS Immortalized Lymphocytes: Towards a Personalized Therapy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition where motor neurons (MNs) degenerate. Most of the ALS cases are sporadic (sALS), whereas 10% are hereditarily transmitted (fALS), among which mutations are found in the gene that codes for the enzyme superoxide dismutase 1 (SOD1). A central question in ALS field is whether causative mutations display selective alterations not found in sALS patients, or they converge on shared molecular pathways. To identify specific and common mechanisms for designing appropriate therapeutic interventions, we focused on the SOD1-mutated (SOD1-ALS) versus sALS patients. Since ALS pathology involves different cell types other than MNs, we generated lymphoblastoid cell lines (LCLs) from sALS and SOD1-ALS patients and healthy donors and investigated whether they show changes in oxidative stress, mitochondrial dysfunction, metabolic disturbances, the antioxidant NRF2 pathway, inflammatory profile, and autophagic flux. Both oxidative phosphorylation and glycolysis appear to be upregulated in lymphoblasts from sALS and SOD1-ALS. Our results indicate significant differences in NRF2/ARE pathway between sALS and SOD1-ALS lymphoblasts. Furthermore, levels of inflammatory cytokines and autophagic flux discriminate between sALS and SOD1-ALS lymphoblasts. Overall, different molecular mechanisms are involved in sALS and SOD1-ALS patients and thus, personalized medicine should be developed for each case.

Progranulin deficiency induces overactivation of WNT5A expression via TNF-α/NF-κB pathway in peripheral cells from frontotemporal dementia-linked granulin mutation carriers.

BACKGROUND: Loss-of-function progranulin gene (GRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with transactive response (TAR) DNA-binding protein 43 (TDP-43) pathology (frontotemporal lobar degeneration [FTLD]-TDP); however, little is known about the association between progranulin (PGRN) deficiency and neuronal loss in individuals with FTLD-TDP. Previously we reported enhanced proliferative activity associated with the activation of WNT5A/CDK6/pRb signalling in PGRN-deficient cells. The objective of this work was to elucidate the association between PGRN deficiency, WNT5A signalling and cell proliferation in immortalized lymphoblasts from carriers of the c.709-1G > A GRN mutation (asymptomatic and FTLD-TDP). METHODS: We assessed cell proliferation in carriers of the c.709-1G > A GRN gene mutation and controls without GRN mutation and without sign of neurologic degeneration by cell counting or using an MTT assay. We used a luciferase assay to measure the nuclear factor-κ (NF-κ) activity. We evaluated messenger RNA levels using quantitative real-time polymerase chain reaction and protein levels by immunoblotting. Co-immunoprecipitation was used to analyze the interaction between PGRN and its receptors. RESULTS: We enrolled 19 carriers of the GRN gene mutation and 10 controls in this study. The PGRN-deficient cells showed increased expression of WNT5A due to NF-κB signalling overactivation. We observed a competition between PGRN and tumour necrosis factor-α (TNF-α) for binding both TNF receptors (TNFR) I and II. Blocking NF-κB signalling using wedelolactone or specific antibodies against TNFRs inhibited WNT5A overexpression and proliferation of PGRN-deficient cells. Conversely, the activation of NF-κB signalling by TNF-α increased WNT5A-dependent proliferation of control cells. LIMITATIONS: All cell lines were derived from individuals harboring the same splicing GRN mutation. Nevertheless, most of the known GRN mutations lead to haploinsufficiency of the protein. CONCLUSION: Our results revealed an important role of NF-κB signalling in PGRN-associated FTLD-TDP and confirm that PGRN can bind to TNF-α receptors regulating the expression of WNT5A, suggesting novel targets for treatment of FTLD-TDP linked to GRN mutations.

Targeting cyclin D3/CDK6 activity for treatment of Parkinson's disease.

At present, treatment for Parkinson's disease (PD) is only symptomatic; therefore, it is important to identify new targets tackling the molecular causes of the disease. We previously found that lymphoblasts from sporadic PD patients display increased activity of the cyclin D3/CDK6/pRb pathway and higher proliferation than control cells. These features were considered systemic manifestations of the disease, as aberrant activation of the cell cycle is involved in neuronal apoptosis. The main goal of this work was to elucidate whether the inhibition of cyclin D3/CDK6-associated kinase activity could be useful in PD treatment. For this purpose, we investigated the effects of two histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic (SAHA) acid and sodium butyrate (NaB), and the m-TOR inhibitor rapamycin on cell viability and cyclin D3/CDK6 activity. Moreover, the potential neuroprotective action of these drugs was evaluated in 6-hydroxy-dopamine (6-OHDA) treated dopaminergic SH-SY5Y cells and primary rat mesencephalic cultures. Here, we report that both compounds normalized the proliferative activity of PD lymphoblasts and reduced the 6-OHDA-induced cell death in neuronal cells by preventing the over-activation of the cyclin D3/CDK6/pRb cascade. Considering that these drugs are already used in clinic for treatment of other diseases with good tolerance, it is plausible that they may serve as novel therapeutic drugs for PD. We report here that peripheral cells from Parkinson's disease (PD) patients show an enhanced proliferative activity due to the activation of cyclin D3/CDK6-mediated phosphorylation of retinoblastoma protein (pRb). Treatment of PD lymphoblasts with inhibitors of histone deacetylases like suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (NaB), or with rapamycin, inhibitor of mechanistic target of rapamycin (mTOR) normalized the proliferation of PD lymphoblasts by preventing the over-activation of the cyclin D3/CDK6/pRb cascade. These drugs were shown to have neuroprotective effects in both human neuroblastoma SH-SY5Y cells and primary rat mid-brain dopaminergic neuronal cultures toxicity induced by 6-hidroxydopamine. Considering that these drugs are already used in clinic for treatment of other diseases with good tolerance, it seems reasonable to believe that the repositioning of these drugs toward PD holds promise as a novel therapeutic strategy.

TTBK1 and CK1 inhibitors restore TDP-43 pathology and avoid disease propagation in lymphoblast from Alzheimer's disease patients.

Introduction: TDP-43 proteinopathy in Alzheimer's disease (AD) patients is recently emerging as a relevant pathomolecular event that may have been overlooked. Recent results in immortalized lymphocytes from AD patients have shown not only an increase of post-translational modifications in TDP-43, such as hyperphosphorylation and fragmentation, but also its prionic behaviour and cell-to-cell disease transmission. With the main goal to advance therapeutic interventions, we present in this work different kinase inhibitors with potential to restore this pathological mechanism. Methodology: We have used immortalized lymphocytes from healthy controls and AD severe patients to evaluate the correction of TDP-43 pathology after the treatment with previously synthetized TTBK1 and CK1 inhibitors. Moreover we used the conditioned mediums of these cells to perform different disease propagation experiments. Results: TDP-43 pathology observed in lymphoblasts from severe AD patients is reduced after the treatment with TTBK1 and CK1 inhibitors (decreasing phosphorylation and increasing nuclear localisation), Furthermore, the significant increase in TDP-43 phosphorylation, cytoplasmic accumulation and aberrant F-actin protrusions (TNT-like structures) observed in control cells growing in CM from AD lymphoblasts were abolished when the CM from AD lymphoblasts treated with previously reported TTBK1 and CK1 inhibitors were used. In addition, the cytosolic transport mediated by molecular motors of the receptor cells was altered with the induced TDP-43 pathology, but it was not produced with the abovementioned pretreated CMs. Conclusion: TTBK1 and CK1 inhibitors, specially VNG1.47 and IGS2.7 compounds, restore TDP-43 pathology and avoid cell-to-cell propagation in immortalized lymphocytes from AD patients, being excellent candidates for the future therapy of this prevalent and devastating disease.

Progranulin Deficiency Induces Mitochondrial Dysfunction in Frontotemporal Lobar Degeneration with TDP-43 Inclusions.

Loss-of-function (LOF) mutations in GRN gene, which encodes progranulin (PGRN), cause frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). FTLD-TDP is one of the most common forms of early onset dementia, but its pathogenesis is not fully understood. Mitochondrial dysfunction has been associated with several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Here, we have investigated whether mitochondrial alterations could also contribute to the pathogenesis of PGRN deficiency-associated FTLD-TDP. Our results showed that PGRN deficiency induced mitochondrial depolarization, increased ROS production and lowered ATP levels in GRN KD SH-SY5Y neuroblastoma cells. Interestingly, lymphoblasts from FTLD-TDP patients carrying a LOF mutation in the GRN gene (c.709-1G > A) also demonstrated mitochondrial depolarization and lower ATP levels. Such mitochondrial damage increased mitochondrial fission to remove dysfunctional mitochondria by mitophagy. Interestingly, PGRN-deficient cells showed elevated mitochondrial mass together with autophagy dysfunction, implying that PGRN deficiency induced the accumulation of damaged mitochondria by blocking its degradation in the lysosomes. Importantly, the treatment with two brain-penetrant CK-1δ inhibitors (IGS-2.7 and IGS-3.27), known for preventing the phosphorylation and cytosolic accumulation of TDP-43, rescued mitochondrial function in PGRN-deficient cells. Taken together, these results suggest that mitochondrial function is impaired in FTLD-TDP associated with LOF GRN mutations and that the TDP-43 pathology linked to PGRN deficiency might be a key mechanism contributing to such mitochondrial dysfunction. Furthermore, our results point to the use of drugs targeting TDP-43 pathology as a promising therapeutic strategy for restoring mitochondrial function in FTLD-TDP and other TDP-43-related diseases.

Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)].

Cumulative evidence indicates that aberrant re-expression of many cell cycle-related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer's disease (AD) pathogenesis. Evidence of cell cycle activation in post-mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway-specific RT(2)Profiler™ PCR Arrays, we detected changes in a number of cell cycle-related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5'-bromo-2'-deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin-dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle-related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis.

TDP-43 Pathology and Prionic Behavior in Human Cellular Models of Alzheimer's Disease Patients.

Alzheimer's disease (AD) is a neurodegenerative disorder for which there is currently no effective treatment. Despite advances in the molecular pathology of the characteristic histopathological markers of the disease (tau protein and ß-amyloid), their translation to the clinic has not provided the expected results. Increasing evidences have demonstrated the presence of aggregates of TDP-43 (TAR DNA binding protein 43) in the postmortem brains of patients diagnosed with AD. The present research is focused on of the study of the pathological role of TDP-43 in AD. For this purpose, immortalized lymphocytes samples from patients diagnosed with different severity of sporadic AD were used and the TDP-43 pathology was analyzed against controls, looking for differences in their fragmentation, phosphorylation and cellular location using Western blot and immunocytochemical techniques. The results revealed an increase in TDP-43 fragmentation, as well as increased phosphorylation and aberrant localization of TDP-43 in the cytosolic compartment of lymphocytes of patients diagnosed with severe AD. Moreover, a fragment of approximately 25 KD was found in the extracellular medium of cells derived from severe AD individuals that seem to have prion-like characteristics. We conclude that TDP-43 plays a key role in AD pathogenesis and its cell to cell propagation.

Multitarget Hybrid Fasudil Derivatives as a New Approach to the Potential Treatment of Amyotrophic Lateral Sclerosis.

Hybrid compounds containing structural fragments of the Rho kinase inhibitor fasudil and the NRF2 inducers caffeic and ferulic acids were designed with the aid of docking and molecular mechanics studies. Following the synthesis of the compounds using a peptide-coupling methodology, they were characterized for their ROCK2 inhibition, radical scavenging, effects on cell viability (MTT assay), and NRF2 induction (luciferase assay). One of the compounds (1d) was selected in view of its good multitarget profile and good tolerability. It was able to induce the NRF2 signature, promoting the expression of the antioxidant response enzymes HO-1 and NQO1, via a KEAP1-dependent mechanism. Analysis of mRNA and protein levels of the NRF2 pathway showed that 1d induced the NRF2 signature in control and SOD1-ALS lymphoblasts but not in sALS, where it was already increased in the basal state. These results show the therapeutic potential of this compound, especially for ALS patients with a SOD1 mutation.

TDP-43 Modulation by Tau-Tubulin Kinase 1 Inhibitors: A New Avenue for Future Amyotrophic Lateral Sclerosis Therapy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease without any effective treatment. Protein TDP-43 is a pathological hallmark of ALS in both sporadic and familiar patients. Post-translational modifications of TDP-43 promote its aggregation in the cytoplasm. Tau-Tubulin kinase (TTBK1) phosphorylates TDP-43 in cellular and animal models; thus, TTBK1 inhibitors emerge as a promising therapeutic strategy for ALS. The design, synthesis, biological evaluation, kinase-ligand complex structure determination, and molecular modeling studies confirmed novel pyrrolopyrimidine derivatives as valuable inhibitors for further development. Moreover, compound 29 revealed good brain penetration in vivo and was able to reduce TDP-43 phosphorylation not only in cell cultures but also in the spinal cord of transgenic TDP-43 mice. A shift to M2 anti-inflammatory microglia was also demonstrated in vivo. Both these activities led to motor neuron preservation in mice, proposing pyrrolopyrimidine 29 as a valuable lead compound for future ALS therapy.

Simvastatin overcomes the resistance to serum withdrawal-induced apoptosis of lymphocytes from Alzheimer's disease patients.

Statins may exert beneficial effects on Alzheimer's disease (AD) patients. Based on the antineoplastic and apoptotic effects of statins in a number of cell types, we hypothesized that statins may be able to protect neurons by controlling the regulation of cell cycle and/or apoptosis. A growing body of evidence indicates that neurodegeneration involves the cell-cycle activation in postmitotic neurons. Failure of cell-cycle control is not restricted to neurons in AD patients, but occurs in peripheral cells as well. For these reasons, we studied the role of simvastatin (SIM) on cell survival/death in lymphoblasts from AD patients. We report here that SIM induces apoptosis in AD lymphoblasts deprived of serum. SIM interacts with PI3K/Akt and ERK1/2 signaling pathways thereby decreasing the serum withdrawal-enhanced levels of the CDK inhibitor p21(Cip1) (p21) and restoring the vulnerability of AD cells to trophic factor deprivation.

CdSe Quantum Dots in Human Models Derived from ALS Patients: Characterization, Nuclear Penetration Studies and Multiplexing.

CdSe quantum dots (QDs) are valuable tools for deciphering molecular mechanisms in cells. Their conjugation with antibodies offers a unique staining source with optimal characteristics, including increased photostability and narrow emission spectra, allowing for improved multiplexing capabilities using a single excitation source. In combination with pathology models derived from patients, they have great potential to contribute to quantitative molecular profiling and promote personalized medicine. However, the commercial availability of diverse CdSe QDs is still limited and characterization techniques must be performed to these materials or the conjugates developed in the lab to assure a proper function and reproducibility. Furthermore, while there is significant data of QDs experiments in cell lines, the literature with primary human cells is scarce, and QD behavior in these systems may be different. Rigorous characterization data of commercially available QDs and their conjugates with biomolecules of interest is needed in order to establish their potential for target labelling and expand their use among research labs. Here we compare the characterization and labelling performance of different QD conjugates in SH-SY5Y cell line, fibroblasts and immortalized lymphocytes derived from amyotrophic lateral sclerosis patients.

Targeting nuclear protein TDP-43 by cell division cycle kinase 7 inhibitors: A new therapeutic approach for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no known cure. Aggregates of the nuclear protein TDP-43 have been recognized as a hallmark of proteinopathy in both familial and sporadic cases of ALS. Post-translational modifications of this protein, include hyperphosphorylation, cause disruption of TDP-43 homeostasis and as a consequence, promotion of its neurotoxicity. Among the kinases involved in these changes, cell division cycle kinase 7 (CDC7) plays an important role by directly phosphorylating TDP-43. In the present manuscript the discovery, synthesis, and optimization of a new family of selective and ATP-competitive CDC7 inhibitors based on 6-mercaptopurine scaffold are described. Moreover, we demonstrate the ability of these inhibitors to reduce TDP-43 phosphorylation in both cell cultures and transgenic animal models such as C. elegans and Prp-hTDP43 (A315T) mice. Altogether, the compounds described here may be useful as versatile tools to explore the role of CDC7 in TDP-43 phosphorylation and also as new drug candidates for the future development of ALS therapies.

A megalin polymorphism associated with promoter activity and Alzheimer's disease risk.

Elevated cerebral levels of amyloid beta-protein (Abeta) occur in Alzheimer's disease (AD), yet only a few patients show evidence of increased Abeta production. This observation suggests that many, perhaps most, cases of AD are caused by faulty clearance of Abeta. Megalin, which plays an important role in mediating Abeta clearance, is an attractive candidate gene for genetic association with AD. To investigate this hypothesis, we analyzed the megalin gene in a population of 2,183 subjects. Genetic analysis indicated that the rs3755166 (G/A) polymorphism located in the megalin promoter associated with risk for AD, dependently of apolipoprotein E genotype. The rs3755166 AA genotype frequency was significantly greater in AD patients than in control subjects. Furthermore, the luciferase reporter assay indicated that the rs3755166 A variant has 20% less transcriptional activity than the rs3755166 G variant. This study provides strong evidence that this megalin polymorphism confers a greater risk for AD, and supports a biological role for megalin in the neurodegenerative processes involved in AD.

Motor neuron preservation and decrease of in vivo TDP-43 phosphorylation by protein CK-1δ kinase inhibitor treatment.

Pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease where no treatment exists, involves the compartmentalization of the nuclear protein TDP-43 (TAR DNA-binding protein 43) in the cytoplasm which is promoted by its aberrant phosphorylation and others posttranslational modifications. Recently, it was reported that CK-1δ (protein casein kinase-1δ) is able to phosphorylate TDP-43. Here, the preclinical efficacy of a benzothiazole-based CK-1δ inhibitor IGS-2.7, both in a TDP-43 (A315T) transgenic mouse and in a human cell-based model of ALS, is shown. Treatment with IGS-2.7 produces a significant preservation of motor neurons in the anterior horn at lumbar level, a decrease in both astroglial and microglial reactivity in this area, and in TDP-43 phosphorylation in spinal cord samples. Furthermore, the recovery of TDP-43 homeostasis (phosphorylation and localization) in a human-based cell model from ALS patients after treatment with IGS-2.7 is also reported. Moreover, we have shown a trend to increase in CK-1δ mRNA in spinal cord and significantly in frontal cortex of sALS cases. All these data show for the first time the in vivo modulation of TDP-43 toxicity by CK-1δ inhibition with IGS-2.7, which may explain the benefits in the preservation of spinal motor neurons and point to the relevance of CK-1δ inhibitors in a future disease-modifying treatment for ALS.