Multiple Roles of Transforming Growth Factor Beta in Amyotrophic Lateral Sclerosis

Transforming growth factor beta (TGFB) is a pleiotropic cytokine, known to be dysregulated in many neurodegenerative disorders and particularly in amyotrophic lateral sclerosis (ALS). This motor neuronal disease is non-cell autonomous, as it affects not only motor neurons, but also their surrounding glial cells, and their target skeletal muscle fibers. Here, we analyze the multiple roles of TGFB in these cell types, and how TGFB signaling is altered in ALS tissues. Data reported support a crucial involvement of TGFB in the etiology and progression of ALS, leading us to hypothesize that an imbalance of TGFB signaling, diminished at the pre-symptomatic stage and then increased with time, could be linked to ALS progression. A reduced stimulation of the TGFB pathway at the beginning of disease blocks its neuroprotective effects and promotes glutamate excitotoxicity. At later disease stages, the persistent activation of the TGFB pathway promotes an excessive microglial activation and strengthens muscular dysfunction. The therapeutic potential of TGFB is discussed here, in order to foster new approaches to treat ALS

Cannabidiolic acid in Hemp Seed Oil Table Spoon and Beyond

Cannabidiolic acid (CBDA) is the main precannabinoid in industrial hemp. It represents a common constituent of hemp seed oil, but mainly abundant in the aerial parts of the plant (including their processing waste). Thus, the optimization of fast and low-cost purification strategies is mandatory, as well as a deep investigation on its nutraceutical and cosmeceutical properties. To this purpose, CBDA content in hemp seed oil is evaluated, and its recovery from wasted leaves is favorably achieved. The cytotoxicity screening towards HaCaT cells, by means of MTT, SRB and LDH release assays, suggested it was not able to decrease cell viability or perturb cell integrity up to 10 μM concentration. Thus, the ability of CBDA to differentially modulate the release of proinflammatory cytokines and chemokines mediators has been evaluated, finding that CBDA decreased IFN-γ, CXCL8, CXCL10, CCL2, CCL4 and CCL5, mostly in a dose-dependent manner, with 10 μM tested concentration exerting the highest activity. These data, together with those from assessing antimicrobial activity against Gram(+) and Gram(-) bacteria and the antibiofilm formation, suggest that CBDA is able to counteract the inflammatory response, also preventing bacteria colonization

VCP mutants cause lysosomal alterations and autophagy induction in ALS-neuronal model

Valosin Containing Protein (VCP) is an ATPase protein that has a key role in various pathways critical for the maintenance of cellular homeostasis and vitality. In particular, VCP is involved in the Protein Quality Control System. Indeed, VCP- mutants have been correlated to different proteinopathies as IBMPFD and ALS. The presence of VCP mutations has been associated with ubiquitin inclusions, TDP-43 mislocalization and aggregation, and abnormal vacuoles. To date VCP-mutants pathological mechanisms are still controversial. Thus, we decided to better define VPC-mutants pathological mechanisms in an ALS-model overexpressing VCP WT, VCP R155H, and VCP R191Q in NSC-34, a motor neuron mouse immortalized cell line. Firstly, we determined that VCP-mutants form insoluble aggregates in this neuronal model. In addition, we observed that the presence of VCP-mutants triggers significant lysosomal alterations in morphology, size, activity, and membrane breakage. Lysosomal alterations have been described to induce cell toxicity and death. To remove damaged lysosomes and therefore to maintain cell vitality, cells activate different mechanisms like autophagy induction. Thus, we analysed LC3 conversion and p62 accumulation, markers of autophagic flux, to determine if the presence of VCP-mutants triggered activation of the autophagic flux. Data showed that VCP- mutants were correlated with an activation of the autophagic flux. Moreover, we determined that the activation of the autophagic flux was specifically regulated by TFE3 calcineurin-dependent dephosphorylation and activation. Calcineurin is a calcium-dependent phosphatase that could be activated by lysosomal leakage supporting a correlation between VCP-mutants lysosomal damage and autophagy activation. In addition, we excluded the involvement of TFEB in this pathway. Together these data suggest that lysosomal damage and leakage induced by VCP- mutants activate calcineurin which in turn mediates TFE3 dephosphorylation and nuclear translocation inducing autophagy. In support of this, we found that VCP- mutants enhanced insoluble protein-aggregates with a specific dependency on the autophagic pathway

Autophagic and proteasomal mediated removal of mutant androgen receptor in muscle models of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease (MND) caused by a mutant androgen receptor (AR) containing an elongated polyglutamine (polyQ) tract. ARpolyQ toxicity is triggered by androgenic AR ligands, which induce aberrant conformations (misfolding) of the ARpolyQ protein that aggregates. Misfolded proteins perturb the protein quality control (PQC) system leading to cell dysfunction and death. Spinal cord motoneurons, dorsal root ganglia neurons and skeletal muscle cells are affected by ARpolyQ toxicity. Here, we found that, in stabilized skeletal myoblasts (s-myoblasts), ARpolyQ formed testosterone-inducible aggregates resistant to NP-40 solubilization; these aggregates did not affect s-myoblasts survival or viability. Both wild type AR and ARpolyQ were processed via proteasome, but ARpolyQ triggered (and it was also cleared via) autophagy. ARpolyQ reduced two pro-autophagic proteins expression (BAG3 and VCP), leading to decreased autophagic response in ARpolyQ s-myoblasts. Overexpression of two components of the chaperone assisted selective autophagy (CASA) complex (BAG3 and HSPB8), enhanced ARpolyQ clearance, while the treatment with the mTOR independent autophagy activator trehalose induced complete ARpolyQ degradation. Thus, trehalose has beneficial effects in SBMA skeletal muscle models even when autophagy is impaired, possibly by stimulating CASA to assist the removal of ARpolyQ misfolded species/aggregates

Enhanced Clearance of Neurotoxic Misfolded Proteins by the Natural Compound Berberine and Its Derivatives

Background: Accumulation of misfolded proteins is a common hallmark of several neurodegenerative disorders (NDs) which results from a failure or an impairment of the proteinquality control (PQC) system. The PQC system is composed by chaperones and the degradative systems (proteasome and autophagy). Mutant proteins that misfold are potentially neurotoxic, thus strategies aimed at preventing their aggregation or at enhancing their clearance are emerging as interesting therapeutic targets for NDs. Methods: We tested the natural alkaloid berberine (BBR) and some derivatives for their capability to enhance misfolded protein clearance in cell models of NDs, evaluating which degradative pathway mediates their action. Results: We found that both BBR and its semisynthetic derivatives promote degradation of mutant androgen receptor (ARpolyQ) causative of spinal and bulbar muscular atrophy, acting mainly via proteasome and preventing ARpolyQ aggregation. Overlapping effects were observed on other misfolded proteins causative of amyotrophic lateral sclerosis, frontotemporal-lobar degeneration or Huntington disease, but with selective and specific action against each different mutant protein. Conclusions: BBR and its analogues induce the clearance of misfolded proteins responsible for NDs, representing potential therapeutic tools to counteract these fatal disorders

Proteotoxic responses in amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease, in which upper and lower motorneurons are differentially affected and are selectively lost during the course of the disease. This results in muscle atrophy and gradually to death of the patients mainly for respiratory failure. ALS mainly occurs as sporadic (s)ALS form. Only 10% of cases are familial (f)ALS; of these, 40% involve the Chromosome 9 Open Reading Frame 72 (C9orf72) gene; other fALS associate to mutations in genes encoding for the proteins SOD1, TDP-43, FUS, SQSTM1/p62, TIA-1, optineurin, ubiquilin, and others, that are causative of motorneuronal death by different pathological mechanisms. One of the most studied is the proteotoxic stress triggered by potentially toxic misfolded proteins that accumulate perturbing several fundamental cell processes. In fact, both as wild type, but particularly when mutated, most of the protein products of these fALS genes (including the aberrant dipeptides (DPRs) arising from unconventional (RAN) translation of the C9ORF72 transcripts) are prone to misfold and to aggregate. The intracellular proteotoxic response to these misfolded proteins and DPRs is based on the coordinate action of chaperones and the degradative systems, mainly the proteasome and the autophagy. Modulators of both the chaperones and of the degradative systems have been found able to modulate misfolded protein toxicity in a variety of preclinical models of fALS. We tested compounds that are able to enhance the autophagy activity and/or to ameliorate the capability of chaperone assisted selective autophagy (CASA) in the removal of ALS associated misfolded proteins including C9ORF72 RAN translated neurotoxic DPRs and their aggregated species. Some of these have been recently clinically tested as potential therapeutic approach to treat different type of neurodegenerative diseases. Fondazione Telethon; Fondazione Cariplo; Fondazione AriSLA; AFM-Telethon, France; Kennedy’s Disease Association, USA; MIUR (PRIN/PNRR; CN3; Dipartimenti Eccellenza); Agenzia Italiana del Farmaco (AIFA)

Motor neuron degeneration in spinal and bulbar muscular atrophy: molecular approaches to counteract mutant androgen receptor neurotoxicity

The neuromuscular disease Spinal bulbar muscular atrophy (SBMA) associates with loss of bulbar or spinal motoneurons and skeletal muscle atrophy. SBMA is caused by a mutation of the androgen receptor (AR) gene resulting in a protein with an elongated polyglutamine (polyQ) tract. ARpolyQ acquires nuclear toxicity after binding testosterone, which induces AR nuclear translocation and ARpolyQ misfolding. Misfolded ARpolyQ is prone to aggregate, a process counteracted by the protein quality control (PQC) system. This system comprises chaperones and the degradative pathways (proteasome and autophagy). Several data suggest that misfolded ARpolyQ is mainly process via autophagy, and causes autophagy flux blockage. Restoration of a functional autophagy is beneficial to cells expressing misfolded ARpolyQ. A peculiar form of autophagy is the "chaperone-assisted selective autoghagy" (CASA), which relies on dynein-mediated retrograde transport of the CASA (HSPB8-BAG3-HSC70-CHIP) complex. This complex binds misfolded ARpolyQ enhancing its clearance. In immortalized motoneurons (MNs) and MNs derived from SBMA iPSCs we found that inhibition of dynein-mediated retrograde transport reduces ARpolyQ accumulation enhancing its clearance. This process is mediated by the HSC70 co-chaperone BAG1 which activate a compensatory mechanism alternative to HSPB8/BAG3. In the knock-in ARQ113 SBMA mouse model (KIARQ113), we found that in affected muscle both BAG1 and BAG3 are upregulated, with an increased BAG3:BAG1 ratio which preferentially routes misfolded ARpolyQ to autophagy. On these basis and on our previous in vitro studies, we tested bicalutamide (an antiandrogen which prevent AR nuclear translocation) and trehalose (an autophagy activator) in KIARQ113 mouse. We found found that mice survival was not significantly modified by the treatments, but an apparent positive trend was present. In Rotarod test KIARQ113 mice the impaired motor coordination was completely recovered by trehalose treatment. Bicalutamide administered at early stages worsened this phenotype (probably because of its anti-anabolic effects on muscle development), but recovered the motor coordination phenotype when administered at later stages (when muscle reached the adulthood stage). Grip strength test in KI mice showed decreased forelimb muscle force, which was further decreased by early, but not late bicalutamide treatment. Hystopathological analyses of mouse gastrocnemius reveal no variation associated to treatments in Feret min and max diameter of the KI mouse muscle fibers. Molecular analyses of gastrocnemius muscle of treated mice showed an increased PGC1alpha expression, paralleled by an increased mitochondrial DNA content and enhanced mitochondrial complex levels (particularly of complex V - ATP synthase subunits and complex III). Thus, the combined trehalose/bicalutamide treatment ameliorates muscle energy production and counteracts ARpolyQ mediated toxicity in vivo

Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration

Macroautophagy/autophagy, a defense mechanism against aberrant stresses, in neurons counteracts aggregate-prone misfolded protein toxicity. Autophagy induction might be beneficial in neurodegenerative diseases (NDs). The natural compound trehalose promotes autophagy via TFEB (transcription factor EB), ameliorating disease phenotype in multiple ND models, but its mechanism is still obscure. We demonstrated that trehalose regulates autophagy by inducing rapid and transient lysosomal enlargement and membrane permeabilization (LMP). This effect correlated with the calcium-dependent phosphatase PPP3/calcineurin activation, TFEB dephosphorylation and nuclear translocation. Trehalose upregulated genes for the TFEB target and regulator Ppargc1a, lysosomal hydrolases and membrane proteins (Ctsb, Gla, Lamp2a, Mcoln1, Tpp1) and several autophagy-related components (Becn1, Atg10, Atg12, Sqstm1/p62, Map1lc3b, Hspb8 and Bag3) mostly in a PPP3- and TFEB-dependent manner. TFEB silencing counteracted the trehalose prodegradative activity on misfolded protein causative of motoneuron diseases. Similar effects were exerted by trehalase-resistant trehalose analogs, melibiose and lactulose. Thus, limited lysosomal damage might induce autophagy, perhaps as a compensatory mechanism, a process that is beneficial to counteract neurodegeneration

Transcriptional induction of the heat shock protein B8 mediates the clearance of misfolded proteins responsible for motor neuron diseases

Neurodegenerative diseases (NDs) are often associated with the presence of misfolded protein inclusions. The chaperone HSPB8 is upregulated in mice, the human brain and muscle structures affected during NDs progression. HSPB8 exerts a potent pro-degradative activity on several misfolded proteins responsible for familial NDs forms. Here, we demonstrated that HSPB8 also counteracts accumulation of aberrantly localized misfolded forms of TDP-43 and its 25 KDa fragment involved in most sporadic cases of Amyotrophic Lateral Sclerosis (sALS) and of Fronto Lateral Temporal Dementia (FLTD). HSPB8 acts with BAG3 and the HSP70/HSC70-CHIP complex enhancing the autophagic removal of misfolded proteins. We performed a high-through put screening (HTS) to find small molecules capable of inducing HSPB8 in neurons for therapeutic purposes. We identified two compounds, colchicine and doxorubicin, that robustly up-regulated HSPB8 expression. Both colchicine and doxorubicin increased the expression of the master regulator of autophagy TFEB, the autophagy linker p62/SQSTM1 and the autophagosome component LC3. In line, both drugs counteracted the accumulation of TDP-43 and TDP-25 misfolded species responsible for motoneuronal death in sALS. Thus, analogs of colchicine and doxorubicin able to induce HSPB8 and with better safety and tolerability may result beneficial in NDs models

The role of the heat shock protein B8 (HSPB8) in motoneuron diseases

Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA) are two motoneuron diseases (MNDs) characterized by aberrant protein behavior in affected cells. In familial ALS (fALS) and in SBMA specific gene mutations lead to the production of neurotoxic proteins or peptides prone to misfold, which then accumulate in form of aggregates. Notably, some of these proteins accumulate into aggregates also in sporadic ALS (sALS) even if not mutated. To prevent proteotoxic stresses detrimental to cells, misfolded and/or aggregated proteins must be rapidly removed by the protein quality control (PQC) system. The small heat shock protein B8 (HSPB8) is a chaperone induced by harmful events, like proteasome inhibition. HSPB8 is expressed both in motoneuron and muscle cells, which are both targets of misfolded protein toxicity in MNDs. In ALS mice models, in presence of the mutant proteins, HSPB8 is upregulated both in spinal cord and muscle. HSPB8 interacts with the HSP70 co-chaperone BAG3 and enhances the degradation of misfolded proteins linked to sALS, or causative of fALS and of SBMA. HSPB8 acts by facilitating autophagy, thereby preventing misfolded protein accumulation in affected cells. BAG3 and BAG1 compete for HSP70-bound clients and target them for disposal to the autophagy or proteasome, respectively. Enhancing the selective targeting of misfolded proteins by HSPB8-BAG3-HSP70 to autophagy may also decrease their delivery to the proteasome by the BAG1-HSP70 complex, thereby limiting possible proteasome overwhelming. Thus, approaches aimed at potentiating HSPB8-BAG3 may contribute to the maintenance of proteostasis and may delay MNDs progression