PhAc-ALGP-Dox, a Novel Anticancer Prodrug with Targeted Activation and Improved Therapeutic Index.

Clinical use of doxorubicin (Dox) is limited by cumulative myelo- and cardiotoxicity. This research focuses on the detailed characterization of PhAc-ALGP-Dox, a targeted tetrapeptide prodrug with a unique dual-step activation mechanism, designed to circumvent Dox-related toxicities and is ready for upcoming clinical investigation. Coupling Dox to a phosphonoacetyl (PhAc)-capped tetrapeptide forms the cell-impermeable, inactive compound, PhAc-ALGP-Dox. After extracellular cleavage by tumor-enriched thimet oligopeptidase-1 (THOP1), a cell-permeable but still biologically inactive dipeptide-conjugate is formed (GP-Dox), which is further processed intracellularly to Dox by fibroblast activation protein-alpha (FAPα) and/or dipeptidyl peptidase-4 (DPP4). In vitro, PhAc-ALGP-Dox is effective in various 2D- and 3D-cancer models, while showing improved safety toward normal epithelium, hematopoietic progenitors, and cardiomyocytes. In vivo, these results translate into a 10-fold higher tolerability and 5-fold greater retention of Dox in the tumor microenvironment compared with the parental drug. PhAc-ALGP-Dox demonstrates 63% to 96% tumor growth inhibition in preclinical models, an 8-fold improvement in efficacy in patient-derived xenograft (PDX) models, and reduced metastatic burden in a murine model of experimental lung metastasis, improving survival by 30%. The current findings highlight the potential clinical benefit of PhAc-ALGP-Dox, a targeted drug-conjugate with broad applicability, favorable tissue biodistribution, significantly improved tolerability, and tumor growth inhibition at primary and metastatic sites in numerous solid tumor models.

Impact of prolonged sepsis on neural and muscular components of muscle contractions in a mouse model.

BACKGROUND: Prolonged critically ill patients frequently develop debilitating muscle weakness that can affect both peripheral nerves and skeletal muscle. In-depth knowledge on the temporal contribution of neural and muscular components to muscle weakness is currently incomplete. METHODS: We used a fluid-resuscitated, antibiotic-treated, parenterally fed murine model of prolonged (5 days) sepsis-induced muscle weakness (caecal ligation and puncture; n = 148). Electromyography (EMG) measurements were performed in two nerve-muscle complexes, combined with histological analysis of neuromuscular junction denervation, axonal degeneration, and demyelination. In situ muscle force measurements distinguished neural from muscular contribution to reduced muscle force generation. In myofibres, imaging and biomechanics were combined to evaluate myofibrillar contractile calcium sensitivity, sarcomere organization, and fibre structural properties. Myosin and actin protein content and titin gene expression were measured on the whole muscle. RESULTS: Five days of sepsis resulted in increased EMG latency (P = 0.006) and decreased EMG amplitude (P < 0.0001) in the dorsal caudal tail nerve-tail complex, whereas only EMG amplitude was affected in the sciatic nerve-gastrocnemius muscle complex (P < 0.0001). Myelin sheath abnormalities (P = 0.2), axonal degeneration (number of axons; P = 0.4), and neuromuscular junction denervation (P = 0.09) were largely absent in response to sepsis, but signs of axonal swelling [higher axon area (P < 0.0001) and g-ratio (P = 0.03)] were observed. A reduction in maximal muscle force was present after indirect nerve stimulation (P = 0.007) and after direct muscle stimulation (P = 0.03). The degree of force reduction was similar with both stimulations (P = 0.2), identifying skeletal muscle, but not peripheral nerves, as the main contributor to muscle weakness. Myofibrillar calcium sensitivity of the contractile apparatus was unaffected by sepsis (P ≥ 0.6), whereas septic myofibres displayed disorganized sarcomeres (P < 0.0001) and altered myofibre axial elasticity (P < 0.0001). Septic myofibres suffered from increased rupturing in a passive stretching protocol (25% more than control myofibres; P = 0.04), which was associated with impaired myofibre active force generation (P = 0.04), linking altered myofibre integrity to function. Sepsis also caused a reduction in muscle titin gene expression (P = 0.04) and myosin and actin protein content (P = 0.05), but not the myosin-to-actin ratio (P = 0.7). CONCLUSIONS: Prolonged sepsis-induced muscle weakness may predominantly be related to a disruption in myofibrillar cytoarchitectural structure, rather than to neural abnormalities.

Restoration of histone acetylation ameliorates disease and metabolic abnormalities in a FUS mouse model.

Dysregulation of epigenetic mechanisms is emerging as a central event in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). In many models of neurodegeneration, global histone acetylation is decreased in the affected neuronal tissues. Histone acetylation is controlled by the antagonistic actions of two protein families -the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). Drugs inhibiting HDAC activity are already used in the clinic as anti-cancer agents. The aim of this study was to explore the therapeutic potential of HDAC inhibition in the context of ALS. We discovered that transgenic mice overexpressing wild-type FUS ("Tg FUS+/+"), which recapitulate many aspects of human ALS, showed reduced global histone acetylation and alterations in metabolic gene expression, resulting in a dysregulated metabolic homeostasis. Chronic treatment of Tg FUS+/+ mice with ACY-738, a potent HDAC inhibitor that can cross the blood-brain barrier, ameliorated the motor phenotype and substantially extended the life span of the Tg FUS+/+ mice. At the molecular level, ACY-738 restored global histone acetylation and metabolic gene expression, thereby re-establishing metabolite levels in the spinal cord. Taken together, our findings link epigenetic alterations to metabolic dysregulation in ALS pathology, and highlight ACY-738 as a potential therapeutic strategy to treat this devastating disease.

HDAC6 as a potential therapeutic target for peripheral nerve disorders.

INTRODUCTION: Peripheral neuropathies are a heterogeneous group of diseases that are characterized by a progressive, ascending loss of nerve function arising from the peripheral regions of the limbs. The phenotypic overlap between different types of hereditary and acquired peripheral neuropathies indicates that similar pathophysiological processes are at play. Many downstream pathways in peripheral neurons, such as axonal transport, protein degradation, and interactions with Schwann cells, organelle damage, channelopathies, and neuroinflammatory signaling, have been proposed and each affects peripheral nerves in a negative way. Histone deacetylase 6 (HDAC6) plays an important role at the intersection of these converging pathogenic pathways. The enzymatic deacetylase activity of HDAC6 is upregulated in neurodegenerative disorders and typically results in downstream neuronal stress. Areas covered: The role of HDAC6 in the common pathogenic mechanisms of peripheral neuropathies. In addition, we discuss the current preclinical and clinical HDAC6 inhibitors (HDAC6i), their chemical structure, development, and limitations. Expert opinion: The development and testing of non-hydroxamic acid-based, should be the focus of the future research. Moreover, HDAC6i should be further investigated as a preventative measure and therapeutic strategy for inherited and acquired peripheral neuropathies.

HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease.

Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.

Testosterone boosts physical activity in male mice via dopaminergic pathways.

Low testosterone (T) in men, especially its free fraction, has been associated with loss of energy. In accordance, orchidectomy (ORX) in rodents results in decreased physical activity. Still, the mechanisms through which T stimulates activity remain mostly obscure. Here, we studied voluntary wheel running behavior in three different mouse models of androgen deficiency: ORX, androgen receptor (AR) knock-out (ARKO) and sex hormone binding globulin (SHBG)-transgenic mice, a novel mouse model of "low free T". Our results clearly show a fast and dramatic action of T stimulating wheel running, which is not explained by its action on muscle, as evidenced by neuromuscular studies and in a muscle-specific conditional ARKO mouse model. The action of T occurs via its free fraction, as shown by the results in SHBG-transgenic mice, and it implies both androgenic and estrogenic pathways. Both gene expression and functional studies indicate that T modulates the in vivo sensitivity to dopamine (DA) agonists. Furthermore, the restoration of wheel running by T is inhibited by treatment with DA antagonists. These findings reveal that the free fraction of T, both via AR and indirectly through aromatization into estrogens, stimulates physical activity behavior in male mice by acting on central DA pathways.

Inhibition of histone deacetylase 6 (HDAC6) protects against vincristine-induced peripheral neuropathies and inhibits tumor growth.

As cancer is becoming more and more a chronic disease, a large proportion of patients is confronted with devastating side effects of certain anti-cancer drugs. The most common neurological complications are painful peripheral neuropathies. Chemotherapeutics that interfere with microtubules, including plant-derived vinca-alkaloids such as vincristine, can cause these chemotherapy-induced peripheral neuropathies (CIPN). Available treatments focus on symptom alleviation and pain reduction rather than prevention of the neuropathy. The aim of this study was to investigate the potential of specific histone deacetylase 6 (HDAC6) inhibitors as a preventive therapy for CIPN using multiple rodent models for vincristine-induced peripheral neuropathies (VIPN). HDAC6 inhibition increased the levels of acetylated α-tubulin in tissues of rodents undergoing vincristine-based chemotherapy, which correlates to a reduced severity of the neurological symptoms, both at the electrophysiological and the behavioral level. Mechanistically, disturbances in axonal transport of mitochondria is considered as an important contributing factor in the pathophysiology of VIPN. As vincristine interferes with the polymerization of microtubules, we investigated whether disturbances in axonal transport could contribute to VIPN. We observed that increasing α-tubulin acetylation through HDAC6 inhibition restores vincristine-induced defects of axonal transport in cultured dorsal root ganglion neurons. Finally, we assured that HDAC6-inhibition offers neuroprotection without interfering with the anti-cancer efficacy of vincristine using a mouse model for acute lymphoblastic leukemia. Taken together, our results emphasize the therapeutic potential of HDAC6 inhibitors with beneficial effects both on vincristine-induced neurotoxicity, as well as on tumor proliferation.

Defective axonal transport: A common pathological mechanism in inherited and acquired peripheral neuropathies.

Peripheral neuropathies are characterized by a progressive and length-dependent loss of peripheral nerve function. This can be caused either by genetic defects, classified as 'inherited peripheral neuropathies', or they can be acquired throughout life. In that case, the disease is caused by various insults such as toxins and mechanical injuries, or it can arise secondary to medical conditions such as metabolic disorders, nutritional deficiencies, inflammation and infections. Peripheral neuropathies are not only very heterogeneous in etiology, but also in their pathology and clinical presentation. A commonality amongst all peripheral neuropathies is that no pharmacological disease-modifying therapies currently exist that can reverse or cure these diseases. Moreover, the length-dependent nature of the disease, affecting the longest nerves at the most distal sites, suggests an important role for disturbances in axonal transport, directly or indirectly linked to alterations in the cytoskeleton. In this review, we will give a systematic overview of the main arguments for the involvement of axonal transport defects in both inherited and acquired peripheral neuropathies. In addition, we will discuss the possible therapeutic strategies that can potentially counteract these disturbances, as this particular pathway might be a promising strategy to find a cure. Since counteracting axonal transport defects could limit the axonal degeneration and could be a driving force for neuronal regeneration, the benefits might be twofold.

Transcriptional upregulation of myelin components in spontaneous myelin basic protein-deficient mice.

Myelin is essential for efficient signal transduction in the nervous system comprising of multiple proteins. The intricacies of the regulation of the formation of myelin, and its components, are not fully understood. Here, we describe the characterization of a novel myelin basic protein (Mbp) mutant mouse, mbp(jive), which spontaneously occurred in our mouse colony. These mice displayed the onset of a shaking gait before 3 weeks of age and seizure onset before 2 months of age. Due to a progressive increase of seizure intensity, mbp(jive) mice experienced premature lethality at around 3 months of age. Mbp mRNA transcript or protein was undetectable and, accordingly, genetic analysis demonstrated a homozygous loss of exons 3 to 6 of Mbp. Peripheral nerve conductance was mostly unimpaired. Additionally, we observed grave structural changes in white matter predominant structures were detected by T1, T2 and diffusion weighted magnetic resonance imaging. We additionally observed that Mbp-deficiency results in an upregulation of Qkl, Mag and Cnp, suggestive of a regulatory feedback mechanism whereby compensatory increases in Qkl have downstream effects on Mag and Cnp. Further research will clarify the role and specifications of this myelin feedback loop, as well as determine its potential role in therapeutic strategies for demyelinating disorders.

Genetic ablation of phospholipase C delta 1 increases survival in SOD1(G93A) mice.

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease, resulting in selective motor neuron degeneration and paralysis. Patients die approximately 3-5 years after diagnosis. Disease pathophysiology is multifactorial, including excitotoxicity, but is not yet fully understood. Genetic analysis has proven fruitful in the past to further understand genes modulating the disease and increase knowledge of disease mechanisms. Here, we revisit a previously performed microsatellite analysis in ALS and focus on another hit, PLCD1, encoding phospholipase C delta 1 (PLCδ1), to investigate its role in ALS. PLCδ1 may contribute to excitotoxicity as it increases inositol 1,4,5-trisphosphate (IP3) formation, which releases calcium from the endoplasmic reticulum through IP3 receptors. We find that expression of PLCδ1 is increased in ALS mouse spinal cord and in neurons from ALS mice. Furthermore, genetic ablation of this protein in ALS mice significantly increases survival, but does not affect astrogliosis, microgliosis, aggregation or the amount of motor neurons at end stage compared to ALS mice with PLCδ1. Interestingly, genetic ablation of PLCδ1 prevents nuclear shrinkage of motor neurons in ALS mice at end stage. These results indicate that PLCD1 contributes to ALS and that PLCδ1 may be a new target for future studies.