A Crucial Role for the Protein Quality Control System in Motor Neuron Diseases.

Motor neuron diseases (MNDs) are fatal diseases characterized by loss of motor neurons in the brain cortex, in the bulbar region, and/or in the anterior horns of the spinal cord. While generally sporadic, inherited forms linked to mutant genes encoding altered RNA/protein products have also been described. Several different mechanisms have been found altered or dysfunctional in MNDs, like the protein quality control (PQC) system. In this review, we will discuss how the PQC system is affected in two MNDs-spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS)-and how this affects the clearance of aberrantly folded proteins, which accumulate in motor neurons, inducing dysfunctions and their death. In addition, we will discuss how the PQC system can be targeted to restore proper cell function, enhancing the survival of affected cells in MNDs

The Regulation of the Small Heat Shock Protein B8 in Misfolding Protein Diseases Causing Motoneuronal and Muscle Cell Death

Misfolding protein diseases are a wide class of disorders in which the aberrantly folded protein aggregates accumulate in affected cells. In the brain and in the skeletal muscle, misfolded protein accumulation induces a variety of cell dysfunctions that frequently lead to cell death. In motoneuron diseases (MNDs), misfolded proteins accumulate primarily in motoneurons, glial cells and/or skeletal muscle cells, altering motor function. The deleterious effects of misfolded proteins can be counteracted by the activity of the protein quality control (PQC) system, composed of chaperone proteins and degradative systems. Here, we focus on a PQC system component: heat shock protein family B (small) member 8 (HSPB8), a chaperone induced by harmful stressful events, including proteotoxicity. In motoneuron and muscle cells, misfolded proteins activate HSPB8 transcription and enhance HSPB8 levels, which contributes to prevent aggregate formation and their harmful effects. HSPB8 acts not only as a chaperone, but also facilitates the autophagy process, to enable the efficient clearance of the misfolded proteins. HSPB8 acts as a dimer bound to the HSP70 co-chaperone BAG3, a scaffold protein that is also capable of binding to HSP70 (associated with the E3-ligase CHIP) and dynein. When this complex is formed, it is transported by dynein to the microtubule organization center (MTOC), where aggresomes are formed. Here, misfolded proteins are engulfed into nascent autophagosomes to be degraded via the chaperone-assisted selective autophagy (CASA). When CASA is insufficient or impaired, HSP70 and CHIP associate with an alternative co-chaperone, BAG1, which routes misfolded proteins to the proteasome for degradation. The finely tuned equilibrium between proteasome and CASA activity is thought to be crucial for maintaining the functional cell homeostasis during proteotoxic stresses, which in turn is essential for cell survival. This fine equilibrium seems to be altered in MNDs, like Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA), contributing to the onset and the progression of disease. Here, we will review how misfolded proteins may affect the PQC system and how the proper activity of this system can be restored by boosting or regulating HSPB8 activity, with the aim to ameliorate disease progression in these two fatal MNDs

Neurodegenerative Disease-Associated TDP-43 Fragments Are Extracellularly Secreted with CASA Complex Proteins

Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control (PQC) system for the cellular clearance of NDs-associated proteins. Here, we investigated the crosstalk between large (LVs) and small (SVs) EVs and PQC in the disposal of TDP-43 and its FTLD and ALS-associated C-terminal fragments (TDP-35 and TDP-25). By taking advantage of neuronal cells (NSC-34 cells), we demonstrated that both EVs types, but particularly LVs, contained TDP-43, TDP-35 and TDP-25. When the PQC system was inhibited, as it occurs in NDs, we found that TDP-35 and TDP-25 secretion via EVs increased. In line with this observation, we specifically detected TDP-35 in EVs derived from plasma of FTLD patients. Moreover, we demonstrated that both neuronal and plasma-derived EVs transported components of the chaperone-assisted selective autophagy (CASA) complex (HSP70, BAG3 and HSPB8). Neuronal EVs also contained the autophagy-related MAP1LC3B-II protein. Notably, we found that, under PQC inhibition, HSPB8, BAG3 and MAP1LC3B-II secretion paralleled that of TDP-43 species. Taken together, our data highlight the role of EVs, particularly of LVs, in the disposal of disease-associated TDP-43 species, and suggest a possible new role for the CASA complex in NDs

RET in breast cancer: pathogenic implications and mechanisms of drug resistance

Initiation, progression, outcome and sensibility to therapies in breast cancer (BC), the most frequent cancer in women, are driven by somatic and germline mutations. Although the effectiveness of hormonal therapies is well-founded, it is prescribed for cancers which express steroid hormone receptors, such as estrogen receptor (ER). RET is a proto-oncogene encoding a transmembrane tyrosine kinase receptor that is activated by one of its four ligands (GDNF, neurturin, artemin or persephin) and one of its coreceptors (Gfrα1-4). Loss-of-function mutations in RET are responsible for Hirschsprung disease, while gain-of-function mutations for multiple endocrine neoplasia type 2. In addition, deregulation of its intracellular signaling, due to mutations, gene rearrangements, overexpression or transcriptional upregulation, can cause several neuroendocrine and epithelial tumors. In BC, amplification of receptor tyrosine kinases, such as ERBB2, EGFR, IGFR and FGFR1, and/or their upregulation contribute to cancer initiation and progression. RET can also have an important role in BC, but only in the subset of ER-positive (ER+) tumors, where it is found overexpressed. Targeting the RET pathway and shedding light on molecular basis of the resistance to hormone therapy may lead to new therapies in ER+ BC, improving treatment outcome and preventing tumor-related events. Thus, here, we review the state of the art of RET biology in BC and agents targeting RET tested in the clinical trials and discuss the specificity of the still available RET inhibitors and the molecular mechanisms underlying the BC resistance to endocrine therapy

Exploration of Tools for the Interpretation of Human Non-Coding Variants

The advent of Whole Genome Sequencing (WGS) broadened the genetic variation detection range, revealing the presence of variants even in non-coding regions of the genome, which would have been missed using targeted approaches. One of the most challenging issues in WGS analysis regards the interpretation of annotated variants. This review focuses on tools suitable for the functional annotation of variants falling into non-coding regions. It couples the description of non-coding genomic areas with the results and performance of existing tools for a functional interpretation of the effect of variants in these regions. Tools were tested in a controlled genomic scenario, representing the ground-truth and allowing us to determine software performance

Valosin Containing Protein (VCP): A Multistep Regulator of Autophagy

Valosin containing protein (VCP) has emerged as a central protein in the regulation of the protein quality control (PQC) system. VCP mutations are causative of multisystem proteinopathies, which include neurodegenerative diseases (NDs), and share various signs of altered proteostasis, mainly associated with autophagy malfunctioning. Autophagy is a complex multistep degradative system essential for the maintenance of cell viability, especially in post-mitotic cells as neurons and differentiated skeletal muscle cells. Interestingly, many studies concerning NDs have focused on autophagy impairment as a pathological mechanism or autophagy activity boosting to rescue the pathological phenotype. The role of VCP in autophagy has been widely debated, but recent findings have defined new mechanisms associated with VCP activity in the regulation of autophagy, showing that VCP is involved in different steps of this pathway. Here we will discuss the multiple activity of VCP in the autophagic pathway underlying its leading role either in physiological or pathological conditions. A better understanding of VCP complexes and mechanisms in regulating autophagy could define the altered mechanisms by which VCP directly or indirectly causes or modulates different human diseases and revealing possible new therapeutic approaches for NDs

The Role of Small Heat Shock Proteins in Protein Misfolding Associated Motoneuron Diseases

Motoneuron diseases (MNDs) are neurodegenerative conditions associated with death of upper and/or lower motoneurons (MNs). Proteostasis alteration is a pathogenic mechanism involved in many MNDs and is due to the excessive presence of misfolded and aggregated proteins. Protein misfolding may be the product of gene mutations, or due to defects in the translation process, or to stress agents; all these conditions may alter the native conformation of proteins making them prone to aggregate. Alternatively, mutations in members of the protein quality control (PQC) system may determine a loss of function of the proteostasis network. This causes an impairment in the capability to handle and remove aberrant or damaged proteins. The PQC system consists of the degradative pathways, which are the autophagy and the proteasome, and a network of chaperones and co-chaperones. Among these components, Heat Shock Protein 70 represents the main factor in substrate triage to folding, refolding, or degradation, and it is assisted in this task by a subclass of the chaperone network, the small heat shock protein (sHSPs/HSPBs) family. HSPBs take part in proteostasis by bridging misfolded and aggregated proteins to the HSP70 machinery and to the degradative pathways, facilitating refolding or clearance of the potentially toxic proteins. Because of its activity against proteostasis alteration, the chaperone system plays a relevant role in the protection against proteotoxicity in MNDs. Here, we discuss the role of HSPBs in MNDs and which HSPBs may represent a valid target for therapeutic purposes

The role of Extracellular Vesicles (EVs) in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD)

ALS and FTLD are neurodegenerative diseases characterized by pathological ubiquitinated and phosphorilated inclusions in the cytosol of affected cells. In 98% of ALS and in the majority of Tau-negative FTLD cases the main component is the TAR DNA-binding protein of 43 KDa (TDP-43) together with its C-terminal fragments of 35 (TDP-35) and 25 KDa (TDP-25). TDP-inclusions are mainly removed from cells via the protein quality control (PQC) system, but they could also be secreted within extracellular vesicles (EVs). In our work we first analysed the TDP-content of the EVs, by comparing large (LVs) with small vesicles (SVs); then, we evaluated the presence of some PQC-members. Finally, we investigated the effect of PQC blockage on EVs secretion and content. Methods. We isolated EVs produced by NSC34 cells untreated or treated with MG132 or NH4Cl (proteasome and autophagy inhibitors). To isolate EVs we used the differential ultracentrifugation method. We analysed EVs size, count and morphology through the Nanoparticle Tracking Analysis and the transmission electron microscopy, and their protein content through western blot analysis. Results. We showed that both TDP-43 and its C-terminal fragments (especially TDP-35) are secreted in EVs, mainly in LVs. Interestingly, in cells TDPs are present as soluble forms, instead the secreted TDPs are mainly insoluble. We found that many PQC-components are secreted in EVs and PQC modulation resulted in a significant increase in EVs numbers, that is paralleled by a slight increase in TDP-content. Summary/Conclusion. EVs may positively contribute to the clearance of insoluble TDPs species by cooperating with PQC, having a protective role for affected cells. However, they may also contribute to the prion-like distribution of TDP-neurotoxic forms in neighboring and more distant cells