Evolving toward a human-cell based and multiscale approach to drug discovery for CNS disorders

A disruptive approach to therapeutic discovery and development is required in order to significantly improve the success rate of drug discovery for central nervous system (CNS) disorders. In this review, we first assess the key factors contributing to the frequent clinical failures for novel drugs. Second, we discuss cancer translational research paradigms that addressed key issues in drug discovery and development and have resulted in delivering drugs with significantly improved outcomes for patients. Finally, we discuss two emerging technologies that could improve the success rate of CNS therapies: human induced pluripotent stem cell (hiPSC)-based studies and multiscale biology models. Coincident with advances in cellular technologies that enable the generation of hiPSCs directly from patient blood or skin cells, together with methods to differentiate these hiPSC lines into specific neural cell types relevant to neurological disease, it is also now possible to combine data from large-scale forward genetics and post-mortem global epigenetic and expression studies in order to generate novel predictive models. The application of systems biology approaches to account for the multiscale nature of different data types, from genetic to molecular and cellular to clinical, can lead to new insights into human diseases that are emergent properties of biological networks, not the result of changes to single genes. Such studies have demonstrated the heterogeneity in etiological pathways and the need for studies on model systems that are patient-derived and thereby recapitulate neurological disease pathways with higher fidelity. In the context of two common and presumably representative neurological diseases, the neurodegenerative disease Alzheimer’s Disease (AD), and the psychiatric disorder schizophrenia (SZ), we propose the need for, and exemplify the impact of, a multiscale biology approach that can integrate panomic, clinical, imaging, and literature data in order to c

Gain and loss of function of P2X7 receptors: Mechanisms, pharmacology and relevance to diabetic neuropathic pain

Background: Genetic causes of exaggerated or reduced pain sensitivity in humans are well known. Recently, single nucleotide polymorphisms (SNPs) in the gene P2RX7, coding for the ATP-gated ion channel P2X7, have been described that cause gain-of-function (GOF) and loss-of-function (LOF), respectively of this channel. Importantly, P2RX7 SNPs have been associated with more or less severe pain scores in patient suffering of post-mastectomy pain and osteoarthritis. Results: The functional consequences of some P2RX7 SNPs (rs208294 (His155Tyr), rs1718119 (Ala348Thr) and rs3751143 (Glu496Ala)) were studied in recombinant cells in vitro. Our findings suggest a correlation between GOF and LOF of P2X7 and actual channel protein expression. Both channel and pore function for these mutant P2X7 receptors changed in parallel to protein levels. On the other hand, the mutant receptors did not differ in their sensitivity to known P2X7 agonists and antagonists. We further demonstrated that in patients with diabetic peripheral neuropathic pain (DPNP), the presence of the GOF SNPs rs208294 (His155Tyr) and rs1718119 (Ala348Thr) is associated, in females, with higher pain intensity scores. Conclusions: Our present results confirm the physiological relevance of some of the SNPs in the P2RX7 gene and show that the presence of these genetic variants correlates with pain sensitivity also in a diabetic neuropathic pain patient population

Impact of exercise and leucine-enriched protein supplementation on physical function, body composition, and inflammation in pre-frail older adults: a quasi-experimental study

BackgroundExercise and a protein-enriched diet are essential for muscle protein synthesis, cellular growth, mitochondrial function, and immune function. The U.S. Food and Nutrition Board's current guideline on recommended dietary allowance for protein in older adults is 0.8 g/kg per day, which may not be sufficient in vulnerable pre-frail older adults.AimsThis study aimed to evaluate the impact of leucine-enriched protein supplementation with or without exercise over 3 months in pre-frail older adults who consumed ≤1 g/kg/day of protein on improving (i) physical function, (ii) body composition measures, and (iii) inflammatory biomarkers such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α).MethodsA non-randomized cluster quasi-experimental study guided by the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist of 178 pre-frail older adults [112 control, 44 nutrition (Nu), and 22 in the nutrition with exercise (Nu+Ex) group] comparing the effect of Nu+Ex and Nu on physical function, body composition, and inflammation. At 0, 3, and 6 months, questionnaires on demographics, depression, perceived health, and cognition were administered. Physical function assessment (short physical performance battery [SPPB] test, gait speed, handgrip strength, 5× sit-to-stand [STS]) was conducted, and body composition analysis was performed using a bioelectrical impedance analysis machine. IL-6 and TNF-α were measured at 0 and 3 months.ResultsAt 3 months, there were significant improvements in gait speed, 5× STS, SPPB scores, depression, perceived health, fat-free mass, and appendicular skeletal muscle mass indices in the Nu+Ex group. Both Nu+Ex and Nu groups had improvements in body cell mass and reductions in IL-6 and TNF-α. The improvements were not sustained after 6 months.ConclusionOur study results need to be validated in future longitudinal randomized studies with a larger sample size focusing on populations at risk

Elevated urine BMP phospholipids in LRRK2 and VPS35 mutation carriers with and without Parkinson's disease

Participant demographic and clinical characteristics, and urine BMP phospholipid levels. For each participant, sample collection site is provided as: BCN (Barcelona), VIE (Vienna), DND (Dundee), or SSB (San Sebastian). Also provided are age at study participation, age at PD diagnosis (where applicable), sex (M, for male, and F, for female), experimental group (control, iPD –idiopathic PD –, LRRK2 G2019S, LRRK2 R1441G/C, VPS35 D620N, GBA, or other), and PD status (NMC for non-manifesting mutation carriers, or PD). Values for all measured BMP species presented as ng of BMP per mg of creatinine are provided. Additionally, urine creatinine (mg/ml) and non-normalized BMP levels are provided. BQL designates BMP levels that were below quantification level and NM designates values that were not measured for a particular individual

The effect of LRRK2 loss-of-function variants in humans

Analysis of large genomic datasets, including gnomAD, reveals that partial LRRK2 loss of function is not strongly associated with diseases, serving as an example of how human genetics can be leveraged for target validation in drug discovery. Human genetic variants predicted to cause loss-of-function of protein-coding genes (pLoF variants) provide natural in vivo models of human gene inactivation and can be valuable indicators of gene function and the potential toxicity of therapeutic inhibitors targeting these genes(1,2). Gain-of-kinase-function variants in LRRK2 are known to significantly increase the risk of Parkinson's disease(3,4), suggesting that inhibition of LRRK2 kinase activity is a promising therapeutic strategy. While preclinical studies in model organisms have raised some on-target toxicity concerns(5-8), the biological consequences of LRRK2 inhibition have not been well characterized in humans. Here, we systematically analyze pLoF variants in LRRK2 observed across 141,456 individuals sequenced in the Genome Aggregation Database (gnomAD)(9), 49,960 exome-sequenced individuals from the UK Biobank and over 4 million participants in the 23andMe genotyped dataset. After stringent variant curation, we identify 1,455 individuals with high-confidence pLoF variants in LRRK2. Experimental validation of three variants, combined with previous work(10), confirmed reduced protein levels in 82.5% of our cohort. We show that heterozygous pLoF variants in LRRK2 reduce LRRK2 protein levels but that these are not strongly associated with any specific phenotype or disease state. Our results demonstrate the value of large-scale genomic databases and phenotyping of human loss-of-function carriers for target validation in drug discovery.Peer reviewe

Characterization of Leishmania donovani MCM4: Expression Patterns and Interaction with PCNA

Events leading to origin firing and fork elongation in eukaryotes involve several proteins which are mostly conserved across the various eukaryotic species. Nuclear DNA replication in trypanosomatids has thus far remained a largely uninvestigated area. While several eukaryotic replication protein orthologs have been annotated, many are missing, suggesting that novel replication mechanisms may apply in this group of organisms. Here, we characterize the expression of Leishmania donovani MCM4, and find that while it broadly resembles other eukaryotes, noteworthy differences exist. MCM4 is constitutively nuclear, signifying that, unlike what is seen in S.cerevisiae, varying subcellular localization of MCM4 is not a mode of replication regulation in Leishmania. Overexpression of MCM4 in Leishmania promastigotes causes progress through S phase faster than usual, implicating a role for MCM4 in the modulation of cell cycle progression. We find for the first time in eukaryotes, an interaction between any of the proteins of the MCM2-7 (MCM4) and PCNA. MCM4 colocalizes with PCNA in S phase cells, in keeping with the MCM2-7 complex being involved not only in replication initiation, but fork elongation as well. Analysis of a LdMCM4 mutant indicates that MCM4 interacts with PCNA via the PIP box motif of MCM4 - perhaps as an integral component of the MCM2-7 complex, although we have no direct evidence that MCM4 harboring a PIP box mutation can still functionally associate with the other members of the MCM2-7 complex- and the PIP box motif is important for cell survival and viability. In Leishmania, MCM4 may possibly help in recruiting PCNA to chromatin, a role assigned to MCM10 in other eukaryotes

PhD

dissertationTo study the functional interactions between neurotensin (NT) and dopamine (DA) systems in the rat brain, the effects of altering dopaminergic activity on the levels of NT-like immunoreactivity (NTLI) in the neostriatum, the nucleus accumbens and the substantia nigra were measured. Dopamine Di receptors were found to help regulate NT systems as methamphetamine (METH)-induced elevations in NTLI contents of these structures were completely blocked by the Di antagonist, SCH 23390. Administration of receptor-selective, direct-acting agonists revealed that Di and D2 receptors have opposite effects on NT systems in both the striatum and the nucleus accumbens. In contrast, stimulation of Di activity by itself did not affect the nigral NT systems, but after activation of D2 receptors a significant increase in the nigral level of NTLI was observed; surprisingly, stimulation of D2 receptors alone reduced the nigral NTLI content. These data suggest that although selective activation of D2 receptors opposes the effect of the Di subtype on nigral NT systems, in combination with Di stimulation, they are facilitatory to nigral Di activity. Elimination of greater than 85% of the central dopaminergic activity completely blocked the elevation in NTLI content caused by METH. For these experiments, depletion of dopamine was achieved by either (i) nigral administration of the dopaminergic neurotoxin, 6-hydroxydopamine, 7 to 10 days before administrations of METH or (ii) multiple doses of reserpine and a-methyl-p-tyrosine. Interestingly, these DA-depleting treatments by themselves induced significant increases in the NTLI content of the striatum and the nucleus accumbens. Although the nigral NTLI content was unaffected by the long-lasting treatments, a single dose of reserpine caused a significant decrease in nigral NTLI content by 18 h after the treatment. These results suggest that NT systems examined are tonically regulated by basal dopaminergic activity, although the nigral response appears to be short-lived. Administration of selective Di or D2 agonists with reserpine showed that tonic dopaminergic regulation of NT systems is mediated primarily by D2 receptors in the striatum and the nucleus accumbens but by Di receptors in the substantia nigra. This was further supported by the observations that interruption of dopaminergic transmission by D2 antagonists (e.g., sulpiride, SULP) increased NTLI levels only in the striatum and the nucleus accumbens whereas the Di antagonist decreased the nigral NTLI content. Finally, another transmitter system(s) appears to be involved in dopaminergic regulation of extrapyramidal NT systems, as destruction of striatal interneurons and/or striatal efferents with the excitotoxin, ibotenic acid, or a knife cut, blocked the METH- and SULP-induced alterations in NT activity of these structures. This linking transmitter system does not appear to be cholinergic since the muscarinic antagonist, atropine, did not attenuate the METH-induced increase in the striatum. However, other studies have shown that glutamatergic projections mediate the effects of METH on NT systems as concurrent blockade of N-methyl-D-aspartate receptors with MK 801 blocked the increases in NTLI contents caused by METH. The SULP-induced alterations, on the other hand, were not blocked by MK 801 suggesting that glutamatergic pathways may not be involved in basal D2 receptor-mediated regulation of NT systems