Therapeutic agents for use in the treatment and prevention of neuropathy, myopathy or cardio(MYO)pathy

Abstract

The invention provides therapeutic agents which can be used in the treatment of neuropathy, myopathy or cardio(myo)pathy. More particularly, the invention provides compounds which are capable of stimulating autophagy, methods for identifying subjects susceptible to treatment with such compounds and methods for identifying such compounds.

Description

[0001] THERAPEUTIC AGENTS FOR USE IN THE TREATMENT AND PREVENTION OF NEUROPATHY, MYOPATHY OR CARDIO(MYO)PATHY

[0002] FIELD

[0003] The invention is broadly in the medical field and provides therapeutic agents which can be used in the treatment of neuropathy, myopathy or cardio(myo)pathy. More particularly, the invention provides compounds which are capable of stimulating autophagy, methods for identifying subjects susceptible to treatment with such compounds and methods for identifying such compounds.

[0004] BACKGROUND

[0005] Protein quality control is important for cellular homeostasis and is ensured by a balance between protein folding and degradation. The heat shock protein (HSP) chaperone system both helps to ensure the native conformation of newly synthesized proteins and to mediate degradation and recycling of denatured proteins in the cell by autophagy. Mutations in the HSPs and other proteins involved in cellular homeostasis have been shown to affect autophagy and have been linked to neuropathy, myopathy and cardiopathy.

[0006] In neuronal tissue, HSPs contribute to the establishment of neuronal proteostasis, supporting the degradation of toxic protein aggregates or damaged cytosolic material. The small heat shock proteins HSPB1 and HSPB8 serve as initial defence against protein aggregation. Mutations in HSPB1 and HSPB8 cause aberrant protein aggregation, alteration of the neuronal structure, and accumulation of axoplasmic material and mitochondria in the muscle and sciatic nerve, resulting in axonal Charcot- Marie-Tooth neuropathies (CMT2), indicating their importance in neuronal homeostasis and the autophagic pathway.

[0007] Similarly, mutations in the Valosin-containin protein VCP has reported impairment in the autophagosome formation leading to the onset of inclusion body myositis with early-onset Paget diseases and frontotemporal dementia (IBMPFD); defective ALS2 / ALSIN and Ubiquilin 2 impair the autophagosome maturation and fusion with consequent neurodegeneration in amyotrophic lateral sclerosis (ALS); mutations in autophagy adaptor proteins like SQSTM1, OPTN, TBK1, ATL1 and ATL3 cause defective autophagy cargo recognition and degradation in ALS, frontotemporal dementia (FTD), Parkinson Disease (PD), hereditary spastic paraplegia (HSP) and hereditary sensory and autonomic neuropathy (HSAN). Deficiencies of LAMP-2, a principal lysosomal membrane protein, have been shown to cause accumulation of intracytoplasmic vacuoles containing autophagic material and glycogen in skeletal and cardiac muscle cells, resulting in vacuolar cardioskeletal myopathy, as seen in Danon's disease. Mutations in Laminin A / C leads to dilated cardiomyopathy with disrupted autophagy flux.

[0008] In light of the direct link between autophagy deficiency and these forms of neuropathy, myopathy and cardiopathy, autophagy stimulation represents a valuable strategy to prevent or treat these disorders.

[0009] The mTOR, the mammalian target of rapamycin, plays a key role in many processes in the cell as it is involved not only in cell growth when nutrients are abundant, but also in the autophagic recycling of cellular components in case of nutrient limitation. mTOR forms two signalling complexes, MTORC1 and mTORC2 by binding with other proteins. Upon nutrient deprivation, mTOR is inactivated and the autophagy cascade is initiated. Pharmacological inhibition of mTORCl, such as by rapamycin, will also induce autophagy.

[0010] The known stimulators of autophagy, such as rapamycin, have however shown to have a detrimental effect on the neuronal network development, induce mitochondrial stress, and neuronal toxicity, making them less suitable for use in the clinic. mTORCl inhibitors approved for cancer treatment, such as everolimus or sirolimus, show major limitations, including severe metabolic and immunosuppressive effects. Therefore, there is a need for novel drugs that trigger autophagy through alternative pathways which could circumvent the side effects of mTOR inhibitors.

[0011] SUMMARY

[0012] The present invention provides therapeutic agents or compounds and compositions for use in the treatment or prevention of a disease or disorder characterized by autophagy dysfunction. In particular embodiments, the disease or disorder is a neuropathy, particularly a peripheral neuropathy, a (distal) myopathy or a cardiopathy, preferably a hereditary peripheral neuropathy, a (distal) myopathy or a cardiopathy (e.g. cardiomyopathy). In particular embodiments, the compounds and compositions are envisaged for use in the prevention of a disorder characterized by an autophagy deficiency in the cells of said subject. In particular embodiments, the subject is characterized by the presence of one or more mutations in genes involved in autophagy. The therapeutic agents of the invention are selected from piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof.

[0013] In particular embodiments, the invention provides for compounds and compositions for use in the treatment or prevention of a neuropathy, a myopathy or a cardiopathy (preferably a cardiomyopathy) in a subject, wherein the subject is characterized by the presence of one or more mutations in a gene selected from the group consisting of Heat Shock Protein Family B Member 1 (HSPB1 / Hsp27; for instance UniProt P04792), Heat Shock Protein Family B Member 3 (HSPB3; for instance UniProt Q12988), Heat Shock Protein Family B Member 5 (HSPB5 / aB-crystallin; for instance UniProt P02511), Heat Shock Protein Family B Member 6 (HSPB6; for instance UniProt 014558), Heat Shock Protein Family B Member 8 (HSPB8 / Hsp22; for instance UniProt Q9UJY1), neurofilament light chain gene (NEFL; for instance UniProt P07196), Ras-related protein Rab-7a (RAB7a; for instance UniProt P51149), Leucine Rich Repeat And Sterile Alpha Motif Containing 1 (LRSAM1; for instance UniProt Q6UWE0), Tripartite Motif Containing 2 (TRIM2; for instance UniProt Q9C040), Beclin-1 (BECN1; for instance UniProt Q14457), Transient Receptor Potential Cation Channel Subfamily V Member 4 (TRVP4; for instance UniProt Q9HBA0), Mitofusin-2 (MFN2; for instance UniProt 095140), Dynamin-2 (DNM2; for instance UniProt P50570), DnaJ heat shock protein family (Hsp40) member B2 (DNAJB2 / HSJ1; for instance UniProt P25686), Member B of the family with sequence similarity 134 (FAM134B / RETREG1; for instance UniProt Q9H6L5), Peripheral myelin protein 22 (PMP22; for instance UniProt Q01453), BAG family molecular chaperone regulator 3 (BAG3; for instance UniProt 095817), transient receptor potential cation channel subfamily V member 4 (TRPV4), VAMP associated protein B (VAPB), WNK lysine deficient protein kinase 1 (WNK1), seipin lipid droplet biogenesis associated (Seipin), ganglioside induced differentiation associated protein 1 (GDAP1), N-myc downstream regulated 1 (NDRG1), tectonin beta-propeller repeat containing 2 (TECPR2), lipopolysaccharide induced TNF factor (LITAF), SH3 domain and tetratricopeptide repeats 2 (SH3TC2), kinesin family member 1A (KIF1A), tyrosine kinase A (TrkA), dynactin 1 (DCTN1), dystonin (DST), valosin containing protein (VCP), phosphoinositide 5-phosphatase (FIG4), myotubularin-related protein 2 (MTMR2), myotubularin- related protein 13 (MTMR13), or chaperonin containing TCP1 subunit 5 (CCT5).

[0014] In particular embodiments, the invention provides for compounds and compositions for use in the treatment or prevention of a neuropathy, a myopathy or a cardiopathy (preferably a cardiomyopathy) in a subject, wherein the subject is characterized by the presence of one or more mutations in a gene selected from the group consisting of peripheral myelin protein 22 (PMP22), transient receptor potential cation channel subfamily V member 4 (TRPV4), leucine rich repeat and sterile alpha motif containing 1 (LRSAM1), VAMP associated protein B (VAPB), WNK lysine deficient protein kinase 1 (WNK1), seipin lipid droplet biogenesis associated (Seipin), DnaJ heat shock protein family (Hsp40) member B2 (DNAJB2), mitofusin 2 (MFN2), ganglioside induced differentiation associated protein 1 (GDAP1), family with sequence similarity 134 member B (FAM134B), N-myc downstream regulated 1 (NDRG1), tectonin beta-propeller repeat containing 2 (TECPR2), lipopolysaccharide induced TNF factor (LITAF), SH3 domain and tetratricopeptide repeats 2 (SH3TC2), kinesin family member 1A (KIF1A), Dynamin 2 (DNM2), tyrosine kinase A (TrkA), dynactin 1 (DCTN1), dystonin (DST), Ras-related GTPase (RAB7), valosin containing protein (VCP), heat shock protein B8 (HSPB8), phosphoinositide 5- phosphatase (FIG4), myotubularin-related protein 2 (MTMR2), myotubularin-related protein 13 (MTMR13), chaperonin containing TCP1 subunit 5 (CCT5), neurofilament light chain gene (NEFL). These genes are involved in or associated with autophagy initiation, autophagosome formation and / or expansion, autophagosome transport, autophagosomal-lysosomal fusion (i.e. autolysosome formation) and / or degradation.

[0015] In particular embodiments, the invention provides for compounds and compositions for use in the treatment or prevention of a neuropathy, a myopathy or a cardiopathy (preferably a cardiomyopathy) in a subject, wherein the subject is characterized by the presence of one or more mutations in a gene selected from the group consisting of Heat Shock Protein Family B Members. In certain embodiments, the Heat Shock Protein Family B Members comprise HSPB1, HSPB3, HSPB5, HSPB6, and HSPB8.

[0016] In particular embodiments, the invention provides for compounds and compositions for use in the treatment or prevention of a neuropathy, a myopathy or a cardiopathy (preferably a cardiomyopathy) in a subject, wherein the subject is characterized by the presence of one or more mutations in a gene selected from the group consisting of Heat Shock Protein Family B Member 1 (HSPB1 / Hsp27), Heat Shock Protein Family B Member 8 (HSPB8 / Hsp22) and / or neurofilament light chain gene (NEFL), Ras- related protein Rab-7a (RAB7a), Leucine Rich Repeat And Sterile Alpha Motif Containing 1 (LRSAM1), Tripartite Motif Containing 2 (TRIM2), Beclin-1 (BECN1), Transient Receptor Potential Cation Channel Subfamily V Member 4 (TRVP4), Heat Shock Protein Family B Member 3 (HSPB3), Mitofusin-2 (MFN2), Dynamin-2 (DNM2), DnaJ heat shock protein family (Hsp40) member B2 (DNAJB2 / HSJ1), Member B of the family with sequence similarity 134 (FAM134B / RETREG1).

[0017] In particular embodiments the subject comprises, in its genome, one or more mutations selected from the Heat Shock Protein Family B Member 1 (HSPB1), Heat Shock Protein Family B Member 8 (HSPB8) and / or neurofilament light chain gene (NEFL).

[0018] In particular embodiments, the mutation is a mutation in the coding sequence of the respective gene. In particular embodiments, the mutation is a mutation which causes an amino acid change in the respective protein (i.e. missense mutation) and / or the mutation is a frameshift mutation or nonsense mutation.

[0019] In particular embodiments, the mutation is a mutation which causes an amino acid change in the HSPB1 protein and / or the HSPB8 protein, preferably in a coding region of the HSPB1 protein and / or the HSPB8 protein. In particular embodiments, the mutation is in the alpha-crystallin domain of the HSPB1 protein, or any other HSPB family member as referred to herein. In particular embodiments, the mutation is a mutation which causes an amino acid change at K141 in the HSPB8 protein, or the corresponding amino acid in any other HSPB family member as referred to herein.

[0020] In particular embodiments, the mutation is a mutation which ensures an amino acid change in the head domain of the NEFL protein, such as, but not limited to in positions P8 or P22 of the NEFL protein.

[0021] In particular embodiments, the compounds and compositions envisaged for use in the context of the present invention comprise one or more of piplartine and pararosaniline pamoate or analogues or derivatives thereof.

[0022] In particular embodiments, the disease or disorder envisaged to be treated or prevented by the therapeutic agent is a hereditary disease. In particular embodiments, the disease or disorder is a peripheral neuropathy, a cardiomyopathy, or a distal myopathy, more particularly a hereditary peripheral neuropathy, a cardiomyopathy, or a distal myopathy.

[0023] In further particular embodiments, the neuropathy is Charcot-Marie-Tooth (CMT) disease, preferably CMT2 disease. In particular embodiments, the CMT2 disease is selected from the following subtypes: patients diagnosed with hereditary motor and sensory neuropathy (HMSN); or patients with predominantly motor involvement, known as distal hereditary motor neuropathy (dHMN), axonal CMT or intermediate subtypes.

[0024] In particular embodiments, the subject envisaged to be treated has been diagnosed to comprise one or more mutations in the genes encoding HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, BAG3, TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5. In particular embodiments, the subject to be treated has been diagnosed with an autophagy deficiency.

[0025] In particular embodiments, the subject envisaged to be treated has been diagnosed to comprise one or more mutations in the genes encoding HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG3. In particular embodiments, the subject to be treated has been diagnosed with an autophagy deficiency.

[0026] In particular embodiments, the subject envisaged to be treated has been diagnosed to comprise one or more mutations in the genes encoding HSPB1, HSPB8 and / or NEFL. In particular embodiments, the subject to be treated has been diagnosed with an autophagy deficiency. In particular embodiments, the therapeutic agent or compound envisaged for use according to the invention is comprised in a pharmacological composition, preferably said pharmacological composition further comprises one or more pharmaceutically acceptable carriers.

[0027] The invention further provides methods for determining the susceptibility of a subject suffering from an autophagy-related disorder to the treatment with piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof, the method comprising, determining whether said subject is characterized by the presence of one or more mutations in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, BAG3, TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5 in its genome.

[0028] The invention further provides methods for determining the susceptibility of a subject suffering from an autophagy-related disorder to the treatment with piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof, the method comprising, determining whether said subject is characterized by the presence of one or more mutations in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PM P22, and / or BAG3 in its genome.

[0029] The invention further provides methods for determining the susceptibility of a subject suffering from an autophagy-related disorder to the treatment with piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof, the method comprising, determining whether said subject is characterized by the presence of one or more mutations in HSPB1, HSPB8 and / or NEFL in its genome.

[0030] The invention further provides methods for determining the susceptibility of a subject suffering from a neuropathy, myopathy or cardio(myo)pathy to the treatment with piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof, the method comprising, determining whether said subject is characterized by the presence of one or more mutations in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, BAG3, TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5 in its genome.

[0031] The invention further provides methods for determining the susceptibility of a subject suffering from a neuropathy, myopathy or cardio(myo)pathy to the treatment with piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof, the method comprising, determining whether said subject is characterized by the presence of one or more mutations in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG3 in its genome.

[0032] The invention further provides methods for determining the susceptibility of a subject suffering from a neuropathy, myopathy or cardio(myo)pathy to the treatment with piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof, the method comprising, determining whether said subject is characterized by the presence of one or more mutations in HSPB1, HSPB8 and / or NEFL in its genome.

[0033] The invention further provides methods for determining the susceptibility of a subject suffering from a neuropathy, myopathy or cardio(myo)pathy to the treatment o with piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof, the method comprising, determining whether said subject is characterized by autophagy deficiency.

[0034] The invention further provides in vitro methods for determining the suitability of an agent for the treatment and / or prevention of an autophagy-related disease or disorder, comprising the steps of: a) determining whether a test agent is capable of rescuing an autophagy deficiency in a cell; and b) comparing the autophagy-rescuing ability of said test agent as determined in a) to the autophagyrescuing ability of piplartine, pararosaniline pamoate or an analogue, derivative or prodrug thereof in said cell.

[0035] BRIEF DESCRIPTION OF DRAWINGS

[0036] Fig. 1 Primary screening identifies hits able to rescue the LC3 puncta count in the Hspb8K141N / K141NMEFs. (a) Scatter plot of the primary screening [in DMSO (negative control; circle), 1 pM Z36 (positive control; square), 1 pM MicroSource (MS) compounds (diamond)]. Each data point represents the average LC3 puncta count per well replicate as percentage to control (POC). The dot dash line indicates the threshold for LC3 puncta count (POC) = 300%, the dashed line indicates the threshold for LC3 puncta count (POC) = 150%. (b) Filtering criteria used to select the list of primary hits. Classification of the filtered hits in two groups according to the potency of the LC3 stimulation. The "non-hits" indicates molecules excluded due to artifacts or limited translation potential.

[0037] Fig. 2 Pararosaniline pamoate and piplartine induce autophagy in the HSPB1_P182L and HSPB8_K141N iPSC-derived motor neurons, (a) Experimental design used for the validation of MC2 (= pararosaniline pamoate) and MC7 (= piplartine) on the iPSC-derived motor neurons (iMN). The top hit compounds MC2 and MC7 were tested in two different concentrations on two healthy control lines and two CMT patient-derived lines. Briefly, motor neuron progenitors (MNP) were seeded (Day 10), at Day 11 the first phase-contrast acquisition was taken to correct for the seeding density, from Day 14 until Day 30 the motor neurons were exposed to the drug treatment. On Day 30, the acute 0.13 pM rapamycin / 10 nM BafAl treatment was applied (data not shown), sample for western blot (WB) were collected, stained with MitoTracker™ Red or fixed for immunocytochemistry (ICC), (b) Western blot on the soluble and insoluble fraction isolated from the iMN (Day 30) after treatment with MC2 or MC7 in two different concentrations (0.13 and 0.25 pM). Samples were blotted for the autophagy marker ATG9A, SQSTMl / p62 and LC3 to monitor the autophagy induction in the soluble fraction. Insoluble protein samples were blotted for SQSTMl / p62 and ubiquitinated protein, (c) Quantification of the LC3 puncta intensity on the neurites of iPSC-derived motor neurons (iMN). Each data point represents an analyzed image (two independent biological replicates, n = 36), outliers removed with ROUT (Q = 5%), two-way ANOVA followed by Dunnett's multiple comparison test, * P < 0.05, ** P < 0.01. (d) Representative confocal images of the LC3B immunostaining of the HSPB1_P182L and HSPB8_K141N mutations under the different treatment conditions (Day 30), red-green lookup table for the LC3 fluorescence intensity, scale bar = 10 pm.

[0038] Fig. 3 Pararosaniline pamoate and piplartine ameliorate the neuronal defects in HSPB1 and HSPB8 iPSC-derived motor neurons. The HSPB1_P182L and the HSPB8_K141N iPSC-derived motor neurons (iMN) were exposed at two different concentrations of pararosaniline pamoate (= MC2) and piplartine (= MC7) during the entire differentiation. Neuronal blebbing, mitochondrial morphology and neurite network area were quantified, (a) Representative confocal images of the tubulin immunostaining performed on the iMN (Day 30) after treatment with MC2 or MC7. Arrowheads indicate neuronal blebbing. Scale bar =10 pm. (b-c) Quantification of the neuronal blebbing upon treatment with MC2 or MC7 in the HSPB1_P182L (b) and HSPB8_K141N (c) reported as neuronal blebbing area and blebbing count, normalized to the average of DMSO-treated neurons. Each data point represents an analyzed image and data are reported as mean ± SEM (data pooled from two independent biological replicates, n=36), outliers removed with ROUT (Q = 5%), One-way ANOVA with Dunnett's multiple comparisons test, (d-e) Quantification of the mitochondrial aspect ratio and mitochondria average size in the HSPB1_P182L (d) and HSPB8_K141N (e). Each data point represents the average per analyzed image (two independent biological replicates, n = 36), outliers removed with ROUT (Q=5%), One-way ANOVA followed by Dunnett's multiple comparisons test, (f-g) Quantification of the neurite area of the HSPB1_P182L and HSPB8_K141N iMN from the phase-contrast pictures obtained after every media change during the MC2 or MC7 treatment. DMSO treatment is indicated in the graph by the lowest line n°l, followed by 0.13 pM (line n°2 and 4) and 0.25pM (lines n°3 and 5) treatments with respectively MC2 (pararosaniline pamoate) and MC7 (piplartine). Dashed lines indicate the start and the end of the treatment, for each time point values are normalized by the seeding density at Day 11, data are reported as mean ± SEM (two independent biological replicates, n = 34). Two-way ANOVA with Dunnett's multiple comparisons test, (h) Representative phase-contrast images of the mutant lines treated with 0.25 pM MC7 (piplartine) resulting in the best neuronal phenotype amelioration compared to the DMSO, scale bar = 50 pm. *P < 0.05 **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0039] Fig. 4 Principal Component Analysis (PCA) identifies two clusters of chemically similar molecules. Using the SMILE, the entire Microsourse library chemical space is represented as simplified 2D space. A proximity score is assigned to each molecule in relation to the other molecules in the library. Colored dots indicate the hits identified in the primary screening (from Table 3). In the circles are highlighted two clusters of chemically similar small molecules identified in the primary screening.

[0040] Fig. 5 Pararosaniline pamoate and piplartine preserve the autophagy flux. AP1= Z36, AP2 = Go6850, MC2 = pararosaniline pamoate, MC7 = piplartine. (a) Dose-response curve of the selected four molecules from the group of potent (Hit group A) and mild autophagy inducers (Hit group B) on the wild-type Hspb8+ / +and mutant Hspb8K141N / K141NMEF clones. Quantification of the cell count (POC) in a range between 0,25 pM and 8 pM. Data are shown as mean ± SEM (n = 3). (b-c) Contribution of the hit compound treatment on the autophagy induction, data are reported as ratio between the Rap / BafAl-stimuated cells and unstimulated cells of the LC3 ratio, respectively for the Hit group A (b) and Hit group B (c). (d-e) ATG9A quantification in the soluble protein fraction isolated from Hspb8K141 / K141NMEFs after treatment with the Hit group A (d) and Hit group B (e) (quantification of the band at 60 kDa). Relative optical densities were normalized to the loading control (P-actin) and reported as mean ± SEM (n = 3 independent biological replicates), (f) Hspb8K141N / K141NMEFs were treated for 4 h with DMSO (vehicle), positive controls (API and AP2) and two hit compounds (MC2 and MC7) with or without 10 nM BafAl to measure the lysosomal degradation. Western blot of the ATG9A, SQSTMl / p62 and LC3. (g) LC3II relative optical densities were normalized to the loading control (P-actin) and reported as mean ± SEM (n=4 independent biological replicates). One-way ANOVA with Tukey's multiple comparison test, (h) Representative TEM images of the autophagic vacuoles (arrowhead) identified in the Hspb8K141N / K141NMEFs treated for 4 h with DMSO, MC2 or MC7 (1 pM) with BafAl 10 nM. *: mitochondrion, #: lysosome, N: nucleus, scale bar = 500 pm.

[0041] Fig. 6 Determination of the top 2 optimal concentrations for the validation on the motor neurons.

[0042] The NEFL_P8R iPSC-derived motor neurons (Pl in the legend) were exposed to a concentration gradient from 0.13 pM to 2 pM during the entire differentiation to determine the optimal concentration for the drug validation. API = Z36, AP2 = Go6850 MC2 = pararosaniline pamoate, MC7 = piplartine (a-d) Quantification of the neurite area as thick neurite bundles (left) and thin neurites (right) from the phase-contrast pictures obtained after every media change during the differentiation and treatment. For each time point, values were normalized by the soma area at Day 11 (24 h post seeding). Dashed lines indicate the start and the end of the treatment. Data are reported as mean values for each tested concentration and timepoint (n = 9). (e) Bundle area fold change at Day 38 compared to the beginning of the differentiation. Data are reported as mean ± SEM (n=9). One-way ANOVA with Holm-Si'ak's multiple comparisons test correction, *P < 0.05, ****p < 0.0001.

[0043] DESCRIPTION

[0044] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0045] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass "consisting of" and "consisting essentially of", which enjoy well-established meanings in patent terminology.

[0046] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0047] The terms "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

[0048] Whereas the terms "one or more" or "at least one", such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more. The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0049] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0050] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

[0051] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0052] Reference throughout this specification to "one embodiment", "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment but may refer to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination. In addition to those detailed herein, the following abbreviations may be used in the present application: Alzheimer's diseases (AD), Amyotrophic lateral sclerosis (ALS), AuTophaGy related (ATG), BAG family molecular chaperone regulator 3 (BAG3), Apoptosis regulator Bcl-2 (BCL2), Chaperone- assisted selective autophagy (CASA), Charcot-Marie-Tooth disease (CMT), Dore-response curve (DRC), Earle's Balanced salt solution (EBSS), European Medicines Agency (EMA); Food and Drug Administration (FDA), Green fluorescent protein (GFP); Gap junction beta-1 protein (GJB1), Histone deacetylase (HDAC), Hereditary motor neuropathy (HMN); Heat shock protein 27 (HSP27), small Heat Shock protein family B (HSPBs), iPSC-derived motor neurons (iMN), Induced Pluripotent Stem Cells (iPSC), Microtubule-associated proteins 1A / 1B light chain 3B (MAP1LC3B (LC3)), Maximum Common Substructure (MCS) similarity (MCSS), Mouse Embryonic Fibroblasts (MEFs), Maximum intensity projection (MIP), Motor Neural Progenitor (MNP), Myelin protein zero (MPZ), mammalian target of rapamycin (mTOR), mammalian target of rapamycin complex 1 / 2 (mTORCl / 2), Neighbor of BRCA1 gene 1 (NBR1), Neurofilament light chain (NEFL), Phagophore assembly site (PAS), Principal component analysis (PCA), Parkinson's disease (PD), Protein kinase C (PKC), Polo-like kinase inhibitor (PLKi), Peripheral myelin protein 22 (PMP22), Peripheral nervous system (PNS), Percent to control (POC), Simplified molecular-input line-entry system (SMILE), Sequestosome 1 (SQSTMl / p62), WD repeat protein interacting with phosphoinositides (WIPI).

[0053] The present inventors have identified therapeutic agents which can rescue autophagy deficiencies caused by a dysfunction of proteins involved in cellular homeostasis, such as caused by mutations in autophagy associated genes, such as those described herein elsewhere. The experiments demonstrate the ability of these compounds to improve the phenotype of cells with a mutant genotype supporting their therapeutic potential.

[0054] The present invention thus provides therapeutic agents or compounds and compositions for use in the treatment or prevention of a disease or disorder characterized by autophagy dysfunction. Autophagy is the natural process that ensures the removal of unnecessary or dysfunctional components in a cell whereby some of the components are recycled for further use in the cell. The process is lysosome-dependent and is essential for cell homeostasis. Autophagy dysfunction in a cell or a subject can occur at the level of control of the process and / or at the level of the individual steps of autophagy including but not limited to vesicle formation, cargo recognition, autophagosome / lysosome fusion, degradation of the cargo. Dysfunction of autophagy occurs when one or more proteins involved in the normal process of autophagy is mutated causing either a decrease or increase of the amount and / or activity of said protein. Accordingly, in certain embodiments, a gene involved in autophagy or associated with autophagy as referred to herein is a gene involved in or associated with vesicle formation, cargo recognition, autophagosome / lysosome fusion, and / or degradation of the cargo, such as the genes referred to herein elsewhere. In certain embodiments, a gene involved in autophagy or associated with autophagy as referred to herein is a gene involved in or associated with autophagy initiation, autophagosome formation and / or expansion, autophagosome transport, autophagosomal-lysosomal fusion (i.e. autolysosome formation) and / or degradation, such as the genes referred to herein elsewhere. The genes as referred to herein may cause autophagy deficiency or dysfunction when mutated. Accordingly, the mutations or as referred to herein may be mutations causing autophagy deficiency or dysfunction and / or the mutated genes as referred to herein may comprise one or more mutations causing autophagy deficiency or dysfunction. Autophagy deficiency or dysfunction may cause neuropathy, myopathy or cardiopathy. Autophagy deficiency can be observed at the cellular level, such as by an increased number of autophagosomes in the cell, accumulation of protein aggregates, accumulation of damaged organelles (fragmented mitochondria), enlarged lysosomes, reduction of the autophagy flux, inactivity of key regulator of the autophagic pathway, often leading to cell degeneration.

[0055] In particular embodiments, the disease or disorder envisaged to be treated herein is a neuropathy, particularly a peripheral neuropathy, a (distal) myopathy or a cardiopathy (particularly cardiomyopathy). In particular embodiments, the peripheral neuropathy, a (distal) myopathy or a cardiopathy is characterized by a deficient autophagy in the cells of the subject. In further particular embodiments, the subject is characterized by the presence of one or more mutations in genes involved in autophagy. As autophagy is a complex process, a large number of genes are involved. In particular embodiments, the gene encodes a heat shock protein that acts as chaperone in the transport and degradation of mature proteins. Examples of proteins involved in autophagy for which mutations have been linked to a neuropathy, myopathy or cardio(myo)pathy include, but are not limited to:

[0056] • Heat Shock Protein Family B Member 1 (HSPB1 / Hsp22) linked to neuropathy in Charcot- Marie-Tooth disease (CMT type 2F and distal HMN) (UniProt P04792)

[0057] • Heat Shock Protein Family B Member 8 (HSPB8 / Hsp27) linked to neuropathy in CMT type 2L and distal HMN (UniProt Q9UJY1)

[0058] • neurofilament light chain gene (NEFL) linked to neuropathy in CMT type 2E (UniProt P07196)

[0059] • Ras-related protein Rab-7a (RAB7a), linked to CMT type 2B (UniProt P51149)

[0060] • Leucine Rich Repeat And Sterile Alpha Motif Containing 1 (LRSAM1), linked to CMT type 2P (UniProt Q6UWE0)

[0061] • Tripartite Motif Containing 2 (TRIM2) linked to early-onset axonal neuropathy (CMT type 2R) (UniProt Q9C040) • Beclin-1 (BECN1), linked to cardiomyopathy (UniProt Q14457)

[0062] • Transient Receptor Potential Cation Channel Subfamily V Member 4 (TRVP4) linked to CMT type 2C (UniProt Q9HBA0)

[0063] • Heat shock protein beta-3 (HSPB3) linked to distal HMN type 2C, myopathy (UniProt Q12988)

[0064] • Mitofusin-2 (MFN2) linked to CMT type 2A, CMT Intermediate type B (UniProt 095140)

[0065] • Dynamin-2 (DNM2), linked to autosomal dominant centronuclear myopathy and intermediate and axonal forms of CMT (UniProt P50570)

[0066] • DnaJ heat shock protein family (Hsp40) member B2 (DNAJB2 / HSJ1) linked to autosomal recessive distal hereditary motor neuronopathy (dHMN) (UniProt P25686)

[0067] • Member B of the family with sequence similarity 134 (FAM134B / RETREG1) linked to a form of peripheral neuropathy called HSAN type 2B (UniProt Q9H6L5).

[0068] • Heat Shock Protein Family B Member 5 (HSPB5 / aB-crystallin) linked to cardio(myo)pathy (UniProt P02511)

[0069] • Heat Shock Protein Family B Member 6 (HSPB6) linked to cardiomyopathy (UniProt 014558)

[0070] • Peripheral myelin protein 22 (PMP22) linked to Charcot-Marie-Tooth type 1A and IE (UniProt Q01453)

[0071] • BAG family molecular chaperone regulator 3 (BAG3) linked to cardio(myo)pathy and CMT type 2 (UniProt 095817)

[0072] • transient receptor potential cation channel subfamily V member 4 (TRPV4)

[0073] • VAMP associated protein B (VAPB)

[0074] • WNK lysine deficient protein kinase 1 (WNK1)

[0075] • seipin lipid droplet biogenesis associated (Seipin)

[0076] • ganglioside induced differentiation associated protein 1 (GDAP1)

[0077] • N-myc downstream regulated 1 (NDRG1)

[0078] • tectonin beta-propeller repeat containing 2 (TECPR2)

[0079] • lipopolysaccharide induced TNF factor (LITAF)

[0080] • SH3 domain and tetratricopeptide repeats 2 (SH3TC2)

[0081] • kinesin family member 1A (KIF1A)

[0082] • tyrosine kinase A (TrkA)

[0083] • dynactin 1 (DCTN1)

[0084] • dystonin (DST)

[0085] • valosin containing protein (VCP)

[0086] • phosphoinositide 5-phosphatase (FIG4)

[0087] • myotubularin-related protein 2 (MTMR2) • myotubularin-related protein 13 (MTMR13)

[0088] • chaperonin containing TCP1 subunit 5 (CCT5)

[0089] In certain embodiments, the mutated genes as referred to herein according to the invention comprise one or more mutations as listed in Table A. Table A

[0090] In particular embodiments, the disease is a hereditary disease. In further particular embodiments, the disease or disorder is a hereditary disease characterized in that the subject has one or more mutations in one or more of the genes encoding the proteins listed above. In the context of the present invention the reference to the protein and genes encoding the protein will be used interchangeably. It will be understood that the genes encoding the above listed proteins are also publicly available. In particular embodiments, the one or more mutations in genes involved in autophagy are in a coding region of the genes encoding these proteins. In particular embodiments, the disease or disorder envisaged to be treated or prevented is peripheral neuropathy, such as Charcot-Marie-Tooth disease. Charcot-Marie- Tooth disease (CMT) refers to a clinically and genetically heterogeneous group of neurological disorders in which peripheral nerves are affected. Traditionally, patients have been classified as having either demyelinating CMT due to myelin abnormalities (CMT type 1), axonal CMT with prominent axon degeneration (CMT type 2), or intermediate CMT with overlapping features. While the majority of CMT1 cases is explained for by mutated genes (PMP22, MPZ, GJB1) expressed in the myelinating Schwann cells, the mutated genes in CMT2 also include molecular chaperones involved in autophagy. About 6% of the CMT2 and distal hereditary motor neuropathy (dHMN) cases have been explained by the identification of mutations in three members of the small heat shock protein family B (HSPBs), such as HSPB1 / Hsp27, HSPB3 and HSPB8 / Hsp22. Accordingly, in particular embodiments of the invention, the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, preferably CMT, whereby the subject has one or more mutations in Heat Shock Protein Family B Member 1 (HSPB1), Heat Shock Protein Family B Member 8 (HSPB8) or Heat shock protein beta-3 (HSPB3). Mutations in HSPB1 and HSPB8 can occur all over the coding regions. Accordingly, in particular embodiments the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, preferably CMT, whereby the subject has one or more mutations in a coding region of the HSPB1 protein and / or HSPB8. For HSB1, mutations have been reported particularly in the alphacrystallin part of the protein. In particular embodiments, the one or more mutations are in the alphacrystallin part of the HSPB1 protein. In further particular embodiments, the mutations are in one or more of codon P182, R127, or S135 of HSPB1, such as but not limited to P182L. For HSPB8, in particular embodiments, the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, preferably CMT, whereby the subject has one or more mutations in a coding region of the HSPB8 protein. For HSPB8 many mutations have been reported in the K141 codon. Accordingly, in particular embodiments the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, preferably CMT, whereby the subject has one or more mutations in codon K141 of HSPB8, such as but not limited to K141N.

[0091] In particular embodiments, the disease envisaged to be treated is CMT type 2. In further particular embodiments, the CMT2 disease is selected from the following subtypes: patients diagnosed with hereditary motor and sensory neuropathy (HMSN); or patients with predominantly motor involvement, known as distal hereditary motor neuropathy (dHMN), axonal CMT or intermediate subtypes.

[0092] In particular embodiments, the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, particularly CMT and the subject comprises one or more mutations in one or more of the genes encoding neurofilament light chain gene (NEFL), Ras-related protein Rab-7a (RAB7a), Leucine Rich Repeat And Sterile Alpha Motif Containing 1 (LRSAM1), Transient Receptor Potential Cation Channel Subfamily V Member 4 (TRVP4), Mitofusin-2 (MFN2) and / or Dynamin-2 (DNM2). For NEFL many mutations associated with disease have been reported in the head domain of the NEFL protein. Accordingly, in particular embodiments the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, preferably CMT, whereby the subject has one or more mutations in the head domain of the NEFL protein, such as in the P8 or P22 position.

[0093] In particular embodiments, the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, particularly CMT and the subject comprises one or more mutations in one or more of the genes encoding HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, BAG3, TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5.

[0094] In particular embodiments, the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, particularly CMT and the subject comprises one or more mutations in one or more of the genes encoding HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG3.

[0095] In particular embodiments, the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, particularly CMT type 2, characterized by axonal degeneration, and the subject comprises one or more mutations in Ras-related protein Rab-7a (RAB7a), Leucine Rich Repeat And Sterile Alpha Motif Containing 1 (LRSAM1) and Mitofusin-2 (MFN2). In particular embodiments, the disease or disorder envisaged to be treated or prevented is a cardiopathy (particularly a cardiomyopathy), and the subject comprises one or more mutations in Beclin-1 (BECN1). In particular embodiments, the disease or disorder envisaged to be treated or prevented is an early onset neuropathy, and the subject comprises one or more mutations in Tripartite Motif Containing 2 (TRIM2).

[0096] In particular embodiments, the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, particularly CMT type 2, characterized by axonal degeneration, and the subject comprises one or more mutations in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, BAG3, TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5.

[0097] In particular embodiments, the disease or disorder envisaged to be treated or prevented is a peripheral neuropathy, particularly CMT type 2, characterized by axonal degeneration, and the subject comprises one or more mutations in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG3.

[0098] The term "mutation" as used herein means any base pair change in the nucleic acid sequence whether it changes the protein's structure or function or has no effect compared to wild type sequence. Unless specified, the term "mutation" is generically used to encompass missense mutation, frameshift mutation, insertions, deletions or substitution of one or more nucleic acids.

[0099] The disease or disorder to be treated or prevented using the compounds of the present invention are typically hereditary disorders. In particular embodiments the disease or disorder has been or is likely to be inherited from one or both parents. In further embodiments, the disease has been or is likely to be inherited from one or more grandparents. Examples of hereditary neuropathies include CMT, particularly CMT type 2, and other related neurodegenerative, neuromuscular diseases, and myopathies.

[0100] The term therapeutic agent as used herein refers to an agent suitable for the treatment or prevention of a disease or disorder.

[0101] As used herein the terms "treat" or "treatment" encompass the therapeutic treatment of an already developed disease or condition. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0102] The term "prevention" as used herein is envisaged to refer to prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction. Beneficial or desired clinical results may include, without limitation, delay of the onset of or prevention of one or more symptoms or one or more biological markers. In particular embodiments, the therapeutic agent is envisaged for use in the treatment or prevention of a neuropathy, myopathy or cardiopathy (such as a cardiomyopathy) in a subject as described herein, wherein said subject has been determined to comprise a mutation in one or more genes as described herein. Thus, in particular embodiments, the presence of one or more mutations in one or more genes associated with autophagy has been determined in a sample of said patient. In particular embodiments, the genes are those as described herein above, more particularly the presence of one or more mutations in HSPB1, HSPB8 and / or NEFL proteins has been established. In particular embodiments, the genes are those as described herein above, more particularly the presence of one or more mutations in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG3 proteins has been established. In particular embodiments, the genes are those as described herein above, more particularly the presence of one or more mutations in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, BAG3, TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5 proteins has been established.

[0103] In particular embodiments, the therapeutic agent of the invention is envisaged for use in a subject which has been diagnosed with an autophagy deficiency.

[0104] The therapeutic agent of the present invention comprises a compound characterized in that it is capable of alleviating autophagy deficiency in a cell, more particularly in a cell characterized by the presence of a mutation in an autophagy related gene. The ability of a compound to alleviate or rescue autophagy deficiency in a cell can be tested by the methods such as disclosed in the examples herein, including but not limited to cellular models with mutant autophagy genes. For instance, the ability of the compound to rescue the autophagy defect in cells characterized by one or more mutant heat shock proteins can be tested. In particular embodiment, the activity of a compound can be tested in a cellular model of a given disease or disorder. In particular embodiments, where the disease envisaged to be treated is a neuropathy, such as CMT, more particularly CMT2, the compound is preferably capable of rescuing the autophagy defect in any of the genes referred to herein, such as HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, BAG3, TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5. In particular embodiments, where the disease envisaged to be treated is a neuropathy, such as CMT, more particularly CMT2, the compound is preferably capable of rescuing the autophagy defect in any of the genes referred to herein, such as HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG3, such as in a cell model comprising a mutation as indicated in Table A. Thus, in particular embodiments, where the disease envisaged to be treated is a neuropathy, such as CMT, more particularly CMT2, the compound is preferably capable of rescuing the autophagy defect in mutant HSPB1 and / or HSPB8 cells, such as in a cell model comprising a mutation in HSPB1 in codon 182 and / or a mutation in HSPB8 in codon 141. For example, the activity of the compound can be validated in a cell line carrying the HSPB1_P182L or HSPB8_K141N mutation. In particular embodiments, the activity of the compound is validated in a human cellular model. In further particular embodiments, where the disease to be treated or prevented is a neuropathy, the activity of the compound is preferably validated in human iPSC-derived motor neurons (iMN). In further particular embodiments, where the disease to be treated or prevented is a myopathy, the activity of the compound is validated in a human iPSC-derived myogenic cell. In further particular embodiments, where the disease to be treated or prevented is a cardiopathy (such as a cardiomyopathy), the activity of the compound is preferably validated in a human iPSC- derived cardiomyogenic cell.

[0105] The therapeutic agents or compounds of the present invention are further preferably characterized in that they have limited side-effects. More particularly, the compounds of the present invention preferably have limited effect on the autophagy flux. The effect of a compound on the autophagy flux of a cell can equally be monitored in cellular models, such as by monitoring expression of other autophagy related genes such as the LC3II / I ratio in presence of a lysosomal inhibitor, SQSTMl / p62 and / or ATG proteins (ATG5-12, ATG7, ATG9A...), quantification of the number of autophagosomes and autolysosomes through the use of reporter moieties, or by monitoring the cargo degradation in the autophagosomes (or other methods such as described by Klionsky et al. 2021, 17(l):l-382).

[0106] The therapeutic agents or compounds of the present invention are further preferably characterized in that they are capable of restoring the overall phenotype of the affected cells. This is indicative not only of therapeutic efficiency but also of treatment tolerance. For instance, autophagy-deficient neurons are characterized by an alteration of the neuronal membrane, mitochondrial morphology, and neurite outgrowth. More specifically, neuronal models of autophagy developed a significantly less dense network compared to the healthy controls and numerous blebbing points along the neurites, as a clear sign of neuronal stress. Preferably, the therapeutic agents of the invention are capable of alleviating one or more of these effects, more preferably the therapeutic agents of the invention restore the cellular phenotype to that of healthy control cells. The therapeutic agents or compounds of the present invention are preferably also characterized in that they are non-toxic and have suitable solubility, permeability, metabolic stability and transporter effects. Methods for testing these characteristics are known in the art.

[0107] In particular embodiments, the therapeutic agent is a compound of Table 3 herein or an analogue, derivative or prodrug thereof. In particular embodiments, the therapeutic agent is selected from pararosaniline pamoate, aklavine hydrochloride, bisanhydrorutilantinone, ergocalciferol, celastrol, piplartine or artemisinin or an analogue, derivative or prodrug thereof. In particular embodiments, the therapeutic agent is selected from pararosaniline pamoate, aklavine hydrochloride, celastrol, and piplartine. In particular embodiments, the compound is piplartine or pararosaniline pamoate, or an analogue, derivative or prodrug thereof.

[0108] Piplartine, also known as piperlongumine, is a natural alkaloid isolated from the medicinal plant Piper longum L. that shows anti-inflammatory activity, neuroprotective and more extensively studied anti- tumoral effect. As a natural product this drug was used for many applications, especially as antitumoral, suggesting it has good distribution properties.

[0109] Due to its established therapeutic potential, many derivatives have already been developed. In particular embodiments, the therapeutic agent envisaged herein is a derivative of piplartine such as a phenylmethylenecyclohexenone derivative, phenylallylcyclohexenone derivative (such as those described in WO2020192348A1 , N-tetradecyl-3,4,5-trimethoxyphenylpropanamide described in CN109516926A, piperonamide analogs according to formulas (I) or (II) described in CN104910174A. Pararosaniline pamoate (pubmed chem 71300937) has been developed as a compound against shistosomiasis and other microorganism infections.

[0110] In this context, different salts novel salts comprising the tris(p-amino phenyl)carbonium cation (pararosaniline cation) and an anion of pamoic acid which is 2,2'-dihydroxy-l, l'-dinaph thylmethane- 3,3'-dicarboxylic acid have been developed. Such salts include bispararosaniline pamoate, a salt comprising two moles of the pararosaniline cation to one mole of the divalent pamoate anion, and pararosaniline acid pamoate, a salt comprising one mole of the pararosaniline cation to one mole of the monovalent acid pamoate anion. The inventors have found that pararosaniline pamoate is capable of restoring autophagy deficiency in a cellular model of CMT.

[0111] In a further related aspect, the invention provides methods for determining the susceptibility of a subject suffering from an autophagy-related disease or disorder to the treatment with the therapeutic agent provided herein, the method comprising, determining whether said subject is characterized by the presence of one or more mutations in one or more autophagy, related genes. Examples of such genes and mutations are those described hereinabove, such as those listed in Table A. In particular embodiments, the method comprises determining, in a sample of said subject, the presence of one or more mutations in the HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5 protein, such as but not limited to the specific mutations described herein above. In particular embodiments, the method comprises determining, in a sample of said subject, the presence of one or more mutations in the HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG3 protein, such as but not limited to the specific mutations described herein above. In particular embodiments, the method comprises determining, in a sample of said subject, the presence of one or more mutations in the HSPB1, HSPB8 and / or NEFL protein, such as but not limited to the specific mutations described herein above. Methods for determining the presence of a given mutation in the genome are known to the skilled person and include mutation analysis using DNA sequencing technologies.

[0112] In particular embodiments, the disease or disorder is a Charcot-Marie-Tooth disease, more particularly a CMT- type 2 disease.

[0113] In a further related aspect, the invention provides in vitro methods for determining the suitability of an agent for the treatment and / or prevention of an autophagy-related disease or disorder in a subject, comprising the steps of: a) determining whether a test agent is capable of rescuing an autophagy deficiency in a cell; and b) comparing the autophagy-rescuing ability of said test agent as determined in a) to the autophagyrescuing ability of the compounds of the present invention.

[0114] In particular embodiments, step a) further comprises determining whether the test agent causes toxicity to said cell. For example, if the cell is a neuron, step a) further comprises determining whether the test agent causes neurotoxicity. For example, if the cell is a neuron, step a) further comprises determining whether the test agent causes neuronal degeneration.

[0115] In particular embodiments, the autophagy-related disorder is a hereditary disorder. In further particular embodiments, the autophagy-related disorder is linked to a mutation in an autophagy related gene. Examples of autophagy related genes are those described herein above. In particular embodiments, the disease is Charcot-Marie-Tooth disease. Examples of particular mutations in an autophagy related gene are those described herein above, such as those listed in Table A. In particular embodiments, the method for determining whether a test agent is capable of rescuing autophagy deficiency in a cell makes use of a cellular model of autophagy as described in the examples and hereinabove. By means of example, and without limitation, capability of rescuing of autophagy deficiency can be based on detection of reduction of AKT phosphorylation, in particular AKT Ser473 phosphorylation, detection of a decrease of p(Ser473)AKT :AKT ratio, detection of LC3 degradation, in particular LC3-II degradation, detection of SQSTM1 (p62) degradation, detection of increase in GABARAP puncta, detection of an increase of Beclin-1 phosphorylation, in particular Beclin-1 Ser93 phosphorylation, or detection of an increase of the p(Ser93)Beclin-l:Beclin-l ratio. Detection results can be compared between samples (in which autophagy is impaired) with or without test compound.

[0116] Additional explanation of terms used in the context of the present invention

[0117] The term "polypeptide" as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms "protein" and "polypeptide" may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expression- type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation / sulfurylation, methylation, ubiquitination, citrullination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes into active forms, etc. The term further also includes polypeptide variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and / or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally occurring polypeptide parts that ensue from processing of such full-length polypeptides.

[0118] A polypeptide or protein can be naturally occurring, e.g., present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom. A polypeptide or protein can be recombinant, i.e., produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesised. Without limitation, a polypeptide or protein can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis. The term "nucleic acid" as used herein typically refers to an oligomer or polymer (preferably a linear polymer) of any length composed essentially of nucleotides. A nucleotide unit commonly includes a heterocyclic base, a sugar group, and at least one, e.g. one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and / or 2-deoxyribose common in naturally occurring nucleic acids, or arabinose, 2- deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof. A modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term "nucleic acid" further preferably encompasses DNA, RNA and DNA / RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA / RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature; or can be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and / or partly or entirely, chemically or biochemically synthesised. A "nucleic acid" can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0119] By "encoding" is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides.

[0120] The reference to any polypeptides, proteins or nucleic acids encompasses such polypeptides, proteins or nucleic acids of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and nonhuman mammals, still more preferably of humans.

[0121] A skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be of precursors of the respective peptides, polypeptides, proteins or nucleic acids and may include parts which are processed away from mature molecules. The term "subject" or "patient" as used herein typically and preferably denotes humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, such as, e.g., non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like.

[0122] The term "therapeutically effective amount" as used herein, refers to an amount of therapeutic agent that elicits the biological or medicinal response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. The term "prophylactically effective amount" refers to an amount of the prophylactic agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. Methods are known in the art for determining therapeutically and / or prophylactically effective amounts of the therapeutic or prophylactic agent as described herein.

[0123] The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples.

[0124] EXAMPLES

[0125] Materials and Methods

[0126] GFP-LC3 / Hspb8 mouse breeding and genotyping

[0127] In order to monitor autophagy, the CMT2L knock-in mouse model for the K141N mutation in the Hspb8 exon 2 was crossed with the transgenic GFP-LC3 model, in which the microtubule-associated protein light chain 3 (LC3) is fused with the green fluorescent protein (GFP) (Bouhy D, et al. Acta neuropathologica. 2018;135(l):131-48; Mizushima N. Methods Enzymol. 2009;452:13-23). Both models had C57BL / 6J genetic background. Homozygous GFP-LC3 animals (GFP-LC3tg / tg) were crossed with homozygous wild-type Hspb8 (Hspb8+ / +) or homozygous knock-in Hspb8 mice (Hspb8K141N / K141N) to obtain the Fl generation of heterozygous GFP-LC3tg / 7Hspb8K141N / +or GFP-LC3tg / + / Hspb8+ / +animals. Genotyping of transgenic animals was performed by PCR as previously described. Genomic DNA was isolated from tail biopsies and amplified for sequencing of Hspb8 exon 2 and to assess the zygosity of the GFP-LC3 transgene. In brief, a set of three primers flanking the insertion position in the genome for the GFP-LC3 transgene were combined in the PCR reaction. Primers amplified two fragments of 250 bp or 350 bp that correspond respectively to the transgenic (tg) and wild-type (+) allele. Breeding and dissections were carried out with the approval of the Ethical Committee for Laboratory Animals, 1

[0128] University of Antwerp (approval file n°2019-41). Mice were housed under the care of the Animal Facility Interfaculty Unit, which is accredited by the Association for Assessment and Accreditation of Laboratory Animals.

[0129] MEF isolation and cell culture

[0130] Mouse embryonic fibroblasts (MEFs) were obtained through timed pregnancy of the Fl generation of the GFP-LC3 / Hspb8 (heterozygous) transgenic mice. Breeding cages were set up, the morning after the females were checked for the presence of the copulation plug and separated from the male breeders. After 13.5 days, pregnant females were euthanized with CO2, the uterus was dissected, and embryos were isolated as individual clones. For each clone, the tail was used for genotyping of the Hspb8 exon 2, and for the GFP-LC3 transgene, the head and red organs were removed to ensure a higher number of remaining fibroblasts (and limit other tissue contamination). The embryonic tissue was minced with sterile scissors and cells were dissociated with 0.25% trypsin-EDTA (ThermoFisher Scientific, 11570626). After two to three dissociation steps, the cell suspension was filtered through a 70-pm strainer before being transferred to a T25 flask in DM EM, supplemented with 10% FBS, 1% penicillin / streptomycin, and 1% glutamine. When confluent, primary MEFs were seeded in a 6-well plate at 150.000 cell / ml and immortalized by transfection with the pSV51 plasmid containing the SV40 large T antigen (28) in PEI MAX 40K (Polyscience Europe, 24765-1) and OptiMEM (ThermoFisher Scientific, 10149832). Transfection medium was replaced with fresh media after 6 hours and MEFs were serially split for at least 7 passages to enrich the culture with immortalized cells with a high duplication rate compared to the primary MEFs. At least 3 clones for each GFP-LC3 / Hspb8 genotype were isolated and immortalized for the autophagy assay.

[0131] Generation of wild-type and mutant HSPB8 stable cell lines

[0132] For the generation of the stable cell lines, HEK293T cells were transfected with the packaging (pCMVdR8.91), envelope (pMD2-VSV) and the transfer plasmid pLENTI6 / V5 containing the human HSPB8 ORF (NM_014365) in its wild type or mutant form Lysl41Asn or Lysl41Glu, using the PEI MAX 40K (Polyscience Europe, 24765-1) and OptiMEM (ThermoFisher Scientific, 10149832). 48 h after transfection, the culture medium with the generated viral particles was collected and filtered through a 0.45-pm filter (Millipore, SLHV033RB). The viral particles were applied on HeLa cells previously seeded in 6-well plate at 150.000 cells / ml. Selection of the positively transduced cells was performed starting 24 h after infection with 100 pg / ml blasticidin (InvivoGen, ant-bl-1). Hela cells were grown in MEM (Life Technologies, 11095080) supplemented with 10% FBS (ThermoFisher Scientific, 10270106), L-Glutamine 2mM (ThermoFisher Scientific, 11539876), and Penicillin-Streptomycin lOOU / ml (ThermoFisher Scientific, 11548876). iPSC lines and motor neuron differentiation

[0133] Three patient iPSC lines bearing the NEFL_P8R, HSPB1_P182L, or the HSPB8_K141N mutation and two age-matched unrelated controls were previously generated and described (29). All the iPSC lines were tested negative for Mycoplasma. The iPSC colonies were maintained on Geltrex™ (Gibco, A1413302)- coated plates in Essential 8™ Flex medium (Gibco, A2858501), when confluent, iPSC colonies were passaged using ReLeSR™ (Stemcell Technologies, 100-0483) and the motor neuron differentiation was initiated with the lines at similar passage numbers. At the start of the differentiation (Day 0), iPSC colonies were scraped and left in clumps to promote the embryoid body (EB) formation. The differentiation protocol was based on previously published work (Van Lent J, Verstraelen P, Asselbergh B, Adriaenssens E, Mateiu L, Verbist C, et al. Induced pluripotent stem cell-derived motor neurons of CMT type 2 patients reveal progressive mitochondrial dysfunction. Brain. 2021;144(8):2471-85). For this study, on Day 10 of the differentiation, embryoid bodies (EBs) were dissociated in single cells and plated on Poly-L-ornithine / laminin-coated plates depending on the defined experimental scheme and assay.

[0134] Antibodies

[0135] The following primary antibodies were used through this study: alpaca GFP-Booster Alexa Fluor® 488 (gb2AF488, ChromTek; 1:400 for ICC), mouse monoclonal anti-SQSTMl / p62 (Abeam, ab56416; 1:1000 for WB, 1:500 for ICC), rabbit polyclonal anti-LC3B (Sigma-Aldrich, L7543; 1: 1000 for WB, 1:200 for ICC), rabbit polyclonal anti-ATG9A (Novus Biologicals, NB110-56893; 1:1000 for WB), mouse monoclonal anti-p-actin (Sigma-Aldrich, A5441; 1:5000 for WB), mouse monoclonal anti-a-Tubulin (GeneTex, GTX112141; 1:5000 for WB), rabbit polyclonal anti-GAPDH (GeneTex, GTX100118; 1:5000 for WB), mouse monoclonal anti- III Tubulin (Abeam, ab7751; 1:500 for ICC), mouse monoclonal anti- Ubiquitin (P4D1) (Cell Signaling, 3936;l:1000 for WB).

[0136] Autophagy assay

[0137] To determine the presence of autophagy defect between the wild-type Hspb8+ / +and the mutant Hspb8K141N / K141NMEF cell lines, the number of GFP-LC3 positive vesicles was quantified when cells were exposed to diverse signals that inhibit the kinase activity of the nutrient sensor mTORCl, thus activating the autophagic pathway. Wild-type or mutant Hspb8 MEFs were seeded on a glass-bottom 96-well plate at 10.000 cells / well in triplicate for each genotype and condition. After 24 h, both physiological and pharmacological mTOR inhibition strategies were applied. Cells were exposed individually to Earle's balanced salt solution (EBSS) in 1:1 ratio with DMEM, or DMEM without FBS supplementation (serum starvation), mTORCl inhibitor rapamycin 200 nM (EMD Millipore, 553210), the ATP-competitive mTORCl and mTORC2 inhibitor torinl 1 pM (Merck Millipore, 475991) in DMEM (30, 31). To assess the autophagy flux, the same experimental conditions were combined with 10 nM of the lysosomal inhibitor bafilomycinAl (Enzo Life Sciences, Inc., BML-CM110-0100). After 2 h treatment, cells were fixed with 2% paraformaldehyde (ThermoFisher Scientific, 28906) at room temperature for 15 min.

[0138] Quantification of the LC3 puncta in Mouse Embryonic Fibroblasts and HeLa cells

[0139] Immunostaining was performed according to standard protocol. Fixed cells were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS, ThermoFisher Scientific, 14190250) for 3 min, following blocking in 5% BSA (Sigma-Aldrich, 9048-46-8) diluted in PBS for 1 h, primary anti-SQSTMl antibody was incubated for 1 h in 5% BSA in PBS, followed by secondary antibody Alexa 594 (1:500) and ChromoTek GFP-Booster Alexa Fluor® 488. Nuclear staining was done by using Hoechst 33342 (Life Technologies, H3570). For the LC3 puncta quantification after autophagy assay, images were automatically acquired with Nikon Eclipse TiE microscope, equipped with DS-Qi2 camera, PFS3, 40x Plan Fluor 0.75 NA objective (DIC M N2), Spectra X epifluorescence excitation source, motorized stage (Prior) and using the JOBS module of Nikon Elements software (Nikon Instruments Europe B.V.). Nine random positions per well were scanned for a multichannel image set (1608x1608 pixels, 0.18 pm / pixel) with PS3 hardware autofocus. Image analysis was performed with CellProfiler software (Broad Institute) by the use of custom-optimized pipeline for the segmentation and quantification of LC3 puncta per cell. First, nuclei are segmented from a binary mask obtained by the Hoechst channel signal with adaptive Otsu thresholding method. Next, the cell area was delimited by propagation from the central nuclei using the SQSTMl-stained channel as cellular mask. Segmentation of punctate structure in the LC3 channel was performed using the Otsu thresholding method. The CellProfiler pipeline provided the final quantification of the number of LC3 punctuated structures per cell for each image, along with cellular shape and intensity parameters. Shape parameters (cell area, minimum and maximum radius) and intensity parameters were used to filter out cells that showed aberrant morphological states (damaged cells, apoptotic cells) or cells that were not correctly segmented in the pipeline. The filters used for the cell selection did not exclude more than 1% of the original cell data set. Data were elaborated in R and statistical analysis was performed in GraphPad Prism.

[0140] Pilot screening with known autophagy inducers and inhibitors

[0141] The focused library SCREEN-WELL® Autophagy library (Enzo Life Sciences, Inc cat. BML-2837) which includes 97 compounds with defined autophagy-inducing or -inhibitory activity was used in a pilot screening to test the assay conditions and to identify a molecule suitable to be used as a positive control. As a first step, 5 nl of 10 mM compounds were pre-dispensed in duplicate using Echo 650 Acoustic Liquid Handler (Beckman Coulter, Inc.) on CellCarrier-384 Ultra microplates (Perkin Elmer, 6057300) coated with Gelatin 0,1% (Millipore, ES-006-B). 0,1% DMSO was added in outer wells as negative controls. Subsequently, Hspb8+ / +and Hspb8K141N / K141NMEF clones were plated at a density of 4000 cells / 40 pl / well using ViaFill multi-dispenser (INTEGRA Biosciences AG.) under sterile conditions. Cells were cultured at 37° and 5% CO2 in DMEM supplemented with 10% Fetal Bovine Serum (heat inactivated at 56° for 30' and steri-filtered) and 1% Penicillin-Streptomycin. After 22 h, autophagy was induced by adding 5 pl of a solution containing 200 nM rapamycin (Sigma-Aldrich, 553210-1MG) and 10 nM bafilomycin Al (Enzo Life Technologies, BML CM110-0100). At 24 h post-drug treatment, cells were fixed for 12 min at 24°C by adding methanol-free paraformaldehyde (Pierce™ 16% Formaldehyde (w / v), Methanol-free, Thermofisher) to a final concentration of 3%, then washed twice with PBS and counterstained with Hoechst 33342 (Invitrogen, H3570). All these last steps have been performed with BioTek EL406 Washer Dispenser (BioTek Instruments, Agilent).

[0142] Primary screening and dose-response curve

[0143] In the primary screening, the Spectrum Collection (Microsource Discovery Systems), which includes 2050 FDA / EMA-approved drugs, natural compounds, and molecules in preclinical stages, was tested on Hspb8K141N / K141NMEFs, as described in the pilot screening. Compounds were tested with a final concentration of 1 pM in duplicate, divided in different plates to prevent plate-specific effect. The compound Z36 identified as an active compound in the pilot screening with SCREEN-WELL® Autophagy library (Enzo Life Sciences, Inc.) has been used as positive control at the concentration of 1 pM. Negative control wells were treated with 0,1% DMSO. Hits were validated with a dose-response curve (DRC). Each compound has been tested in both Hspb8+ / +and Hspb8K141N / K141NMEF clones, in three replicates for six concentration points: 0,25 - 0,50 - 1,00 - 2,00 - 4,00 - 8,00 pM using Echo 650 Acoustic Liquid Handler (Beckman Coulter, Inc.). Drug addition, cell seeding, autophagy induction fixation and staining were performed as described in the pilot screening.

[0144] Screening image acquisition and analysis

[0145] Image acquisition was performed using ImageXpress Micro Confocal High Content System (Molecular Devices, LLC). Four images per well were acquired with 20X Plan Apo Lambda 0,75NA Objective in Widefield Mode for two channels: Hoechst - Blue Ex 377 / 54 and Em 432 / 36, GFP-LC3 Green Ex 475 / 28 Em 536 / 40. Images were analyzed using the Custom Module extension of MetaXpress 6.7.1.157 (Molecular Devices, LLC). Nuclei were counted based on the Hoechst staining, the cytosolic region was defined using a low threshold segmentation on the green and blue channels and GFP-LC3 puncta were counted based on the GFP signal in a size range between 0.3 and 3.0 pm2. To define the effect of a compound the total number of cells per well was considered as a proxy of the cell viability, and the mean LC3 puncta per cell was calculated as measure of the autophagy induction. The average GFP intensity was used to filter out autofluorescence artifacts. Statistical analysis was performed using KNIME Analytics Platform 4.5.2. Raw data were intra-plate normalized by the DMSO-treated wells (negative control), expressed as a percentage of control (POC). To assess the quality of the screening, the / 'factor and the CV% were calculated per plate between positive and negative controls.

[0146] Positive control and hit selection

[0147] In the pilot screening, compounds candidate as positive controls were selected considering a low cytotoxic effect to the cells (>80% cell count POC) and an increased number of GFP-LC3 puncta per cell (>120% POC), but also a selectively higher induction of GFP-LC3 puncta in the mutant Hspb8 MEF clone compared to the wild type (fold change >1.2). A DRC approach was applied to the selected list of molecules to identify the most suitable positive control. In the primary screening, compounds with high cytotoxic effect (<50% cell count POC) were excluded from the hit selection together with autofluorescent compounds (average green intensity POC >200%). To evaluate the autophagy induction based on the GFP-LC3 puncta, the filtered compounds were divided into two hits groups: Hit group A with >300% LC3 puncta POC and Hit group B with LC3 puncta POC between 120% and 300%.

[0148] Chemical similarity analysis (Principal component analysis)

[0149] Hit compounds were investigated for their chemical similarity using KNIME Analytics Platform 4.5.2 (33). Briefly, for each library compound, the RDKit molecules was generated starting from the molecule SMILE using the RDKit from Molecule node, then the RDKit Fingerprint node was used to generate the Morgan fingerprint type for each molecule . A Tanimoto index was calculated using the Distance Matrix node. The MoSS MCSS molecule similarity node was used to compute similarities between pairs of molecules by taking the size of their Multiple Copy Similarity Search as a measure for similarity (the bigger the MCSS the more similar the molecules are). For computing the MCSS, the substructure miner MoSS was used. Subsequently the Similarity search node was used to check the similarity of each library molecule with each neighbor based on the distance measures. The fingerprint of each molecule was expanded using the Expand Bit Vector node. The resulting 1024 integer columns were included, together with the neighbor index and the similarity index, in a Principal Component Analysis (PCA). The resulting 6 dimensions were plotted to identify hits compound clusters.

[0150] Immunoblotting The Hspb8K141N / K141NMEF were seeded in duplicate in 6-well plate at 300.000 cells / well. 24 h after seeding cells were exposed to the hit compounds from the primary screening at 1 pM for 22 h with or without exposure to rapamycin 200 nM and bafilomycin 10 nM for 2 h. Cells were collected in ice-cold PBS and pelleted 1 min at 1699 ref. For the iMN, the motor neuron progenitors were seeded in 6-well plates at 250.000 cells / well. At Day 30, the entire developed neurite network is detached with IX PBS. The cell pellet was lysed in RIPA buffer (1% Nonidet P-40, 150 mM NaCI, 0.1% SDS, 0.5% deoxycholic acid, ImM EDTA, 50 mM Tris-HCI, pH 7.5) with complete protease (Roche Applied Science, 4693159001) and Phospho-STOP inhibitor mixtures (Roche Applied Science, 4906837001) for 20 min on ice and cleared by centrifugation for 15 min at 20817 ref. The insoluble RIPA fraction was saved for the iMN to determine the presence of SQSTMl / p62 aggregates or ubiquitinated protein. Soluble proteins in the cell lysate were quantified by Pierce BCA protein assay kit (ThermoFisher Scientific, 23225) and equally loaded on NuPAGE™ 4-12% Bis-Tris gel or NuPAGE™ 12% Bis-Tris gel (ThermoFisher Scientific, 10247002, 12030166) for LC3 isoform detection. Proteins were blotted on nitrocellulose membrane (10600003, Amersham Biosciences Europe GmbH), blocking was performed with 5% milk powder dissolved in PBS supplemented with 0.5% Tween-20 (Sigma Aldrich, P1379). Alternatively, for the detection of phosphorylated proteins, membranes were blocked with 5% Bovine Serum Albumine (BSA) in Tris-buffered saline with 0.1% Tween® 20 detergent (TBS-T). Primary antibodies were incubated for 1 h at room temperature or overnight at 4°C, depending on manufacturer recommendation, and secondary horseradish peroxidase-conjugated antibody for lh at room temperature. Membranes were developed with enhanced chemiluminescence ECL PlusTM detection system (ThermoFisher Scientific, 32134) and imaged with an Amersham 600 Imager operated with ImageQuant software (GE Healthcare Life Sciences) and using the semi-automated exposure program. Exposure time was determined by manually selecting the region of interest with the bands at the correct molecular weight height. The software calculates the appropriate exposure time that will give the highest possible signal below saturation and enables accurate quantification of the sample. The images were then saved in 16-bit image files. Band intensities were determined by quantifying the mean pixel gray values using ImageJ software . Mean pixel gray values were measured in a rectangular region of interest for each protein and normalized by the loading control.

[0151] Transmission electron microscopy

[0152] From a confluent T175 culture flask, cells were resuspended in 10 ml of MEM and divided into 10-cm dished according to the required treatments. The day after seeding, cells were exposed to fresh medium containing 1 pM of the selected hits in combination with bafilomycinAl (10 nM) or with DMSO for 4 h. Next cells were dissociated, collected in 15 ml tubes, and divided for TEM sample preparation and for a parallel western blot confirmation. For transmission electron microscopy (TEM), cells were pelleted for 5 min at 900 rpm in 5 ml tubes (Eppendorf, 0030119487), fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate and 0.05% CaCl2*2H2O, washed three times in 0.1 M sodium cacodylate-buffered (pH 7.4) 7.5 % saccharose solution. Post-fixation was performed by incubating cells for 2 h with 1 % OsO4 solution. After dehydration in an ethanol gradient, samples were embedded in EM-bed812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Tecnai G2 Spirit Bio Twin Microscope (FEI, Eindhoven, The Netherlands) at 120 kV. Representative images were acquired to confirm the presence of autophagic vacuole in the cells. Autophagic vacuoles were defined according to morphological features as described in literature: vesicles delimited by an intact membrane, containing electron-dense material similar to the cytoplasm or containing other membranes and part of organelles, in a range of 300-900 nm. In the case of autophagosomes, the double membrane was visible, however upon fusion with the lysosomes, the autolysosomes lose the internal membrane, and the internal area appears dark, and the content is partially broken down. iPSC-derived motor neuron drug validation

[0153] Top hit validation was performed on iPSC-derived motor neurons in two phases. First, to determine possible neurotoxicity, the optimal dose was defined through a dose-response on the NEFL_P8R iMN, a line that carries one of the most severe CMT mutations, responsible for early onset disease phenotypes (Van Lent J, Verstraelen P, Asselbergh B, Adriaenssens E, Mateiu L, Verbist C, et al. Induced pluripotent stem cell-derived motor neurons of CMT type 2 patients reveal progressive mitochondrial dysfunction. Brain. 2021;144(8):2471-85, Jordanova A, De Jonghe P, Boerkoel CF, Takashima H, De Vriendt E, Ceuterick C, et al. Mutations in the neurofilament light chain gene (NEFL) cause early onset severe Charcot-Marie-Tooth disease. Brain. 2003;126(Pt 3):590-7). Second, the best two concentrations were used on the HSPB1_P182L and HSPB8_K141N lines, along with two healthy control lines. On Day 10 of the differentiation protocol, the embryoid bodies (EBs) were dissociated in single cells and the motor neuron progenitors (MNP) were plated on Poly-L-ornithine / laminin-coated plates depending on the defined experimental scheme and assay. The drug treatment was applied every other day starting from Day 14 to Day 30 by replacing half media, supplemented with the hit compounds. Untreated control samples received complete neuronal medium (NT) or neuronal medium with 0.01% DMSO. As reference control for the canonical autophagy induction, iMN were either exposed to rapamycin 0.13 pM during the entire differentiation (chronic stimulation) or on the day of collection, iMN were exposed to 200 nM rapamycin and 10 nM bafilomycinAl for 3 h (acute stimulation). The work on human iPSC was approved by the local Medical Ethics Committee of the University of Antwerp (file n° CME B300201319371).

[0154] Neurite network analysis

[0155] Motor neuron progenitors were seeded in 24-well plates at 50,000 cells / well. For every medium change during the differentiation from Day 11 to Day 36, plates were placed in the time-lapse microscopy system Incucyte, acclimatized for at least 30 min, and scanned with the 20X objective, nine phase contrast positions in grid layout per well were acquired (1408x1040 pixels, 0.62 pm / pixel). At the end of the differentiation, all the images were exported and analyzed with llastik. A random Forest classifier is trained by annotating pixels to different classes (bundle, neurite, soma, necrotic body, and background) on a set of random images, binary segmentation masks were generated and the number of pixels for each class was quantified by in-house developed Fiji ImageJ macro. The neurite pixel number was normalized by the soma pixels at Day 11 (day in which the progenitors were still single isolated cells) to correct for the seeding density per each well. Data were reported as mean ± SEM for each time point, a continuous hinge function was used to build a segmental regression with gentle connection to interpolate the data and obtain the growth curves.

[0156] Mitochondrial morphology analysis

[0157] Motor neuron progenitors were plated in 96-well Ibidi plates (15000 cells / well). At Day 30, iMN were incubated with 250 nM MitoTracker™ Red FM (ThermoFisher Scientific, M22425) for 30 minutes at 37°C. Afterward, the medium was replaced with fresh neuronal medium to limit background signal and neurons were fixed with 2% PFA for 25 minutes at room temperature. Fixation medium was removed and replaced with PBS. Imaging was performed with the spinning disk confocal Nikon eclipse Ti2 microscope equipped with a kinetic A2B723016 camera, PFS, Plan Apo lambda 40X air objective (MRD00405), NIDAQ. Piezo Z device, using the JOBS module of the NIS-Elements software. Nine random positions (2048x2048 pixel, 0.16 pm / pixel) per well were imaged at 561 nm excitation and 4,8 pm Z-stacks (1,2 pm inter step) were automatically acquired and saved as Maximum Intensity Projection (MIP). The MIPs were used to train a classifier in llastik to discriminate the mitochondria to the background, next, the generated binary mask was used to identify the mitochondria as particles. Shape parameters (minimum and maximum diameter, circularity) were analyzed using the Fiji distribution of ImageJ for each single particle and reported as average per image field.

[0158] Neuronal LC3 puncta and blebbing quantification

[0159] Motor neuron progenitors are seeded in 96-well Ibidi plates (15000 cells / well). At Day 30, mature motor neurons were fixed with 2% PFA directly in the culture medium for 25 min at room temperature and washed once with PBS. Permeabilization was performed with 0.5% Triton™ X-100 (Sigma, T8787) in PBS for 3 min, followed by the addition of antigen-blocking buffer for 1 h at room temperature with 5% Bovine Serum Albumin / 0.1% triton-XlOO dissolved in PBS. Primary antibodies were incubated overnight at 4°C in BSA 5% / IX PBS. Afterward, secondary antibodies DaR-488 (1:500, Life Technologies, A21206) and GaM(Gl)-594 (1:500, Life Technologies, A21125) were incubated for lh at room temperature. Nuclear staining was performed with Hoechst33342 (Life Technologies, H3570) and applied for 3 min. Long-term storage of the stained samples was guaranteed with 0.01% sodium azide / PBS. Images were acquired with the spinning disk confocal Nikon eclipse Ti2 microscope equipped with a kinetic A2B723016 camera, PFS, Plan Apo lambda 40X air objective (NA 0.95, MRD00405), NIDAQ. Piezo Z device, using the JOBS module of the NIS-Elements software. Nine random multichannel positions (2048x2048 pixels, 0.16 pm / pixel calibration) per well were defined and 15 pm Z-stack (1.5 pm inter step) in the three channels were automatically acquired and saved as Maximum Intensity Projection. Next the images are processed with llastik. For each MIP two independent pixel classifiers were generated for the total neurite network or for the LC3-positive particles, making also a distinction between the soma and neurites. Only the LC3-positive pixel within the neurites were considered for the analysis.

[0160] We generated a third binary mask for neuronal blebbing. Neuronal blebbing is defined as membranous circular protrusions along the axons and in proximity of the cell bodies that contain (tubulin-dense) axoplasmic material and is associated with neurotoxicity and axonal degeneration. Through the binary mask, the neuronal blebs were identified as particles in a range between 50-3000 pixels. The number of particles and blebbing area were normalized by the total neuronal network per image.

[0161] Statistical analysis

[0162] For the pilot screening and primary screening, the statistical significance of the positive hits was measured based on the response of each compound as percentage to control (POC). For all the other experiments statistical analysis was performed with GraphPad Prism 10.0.2 software. The data are presented as mean ± SEM. Normality assessment was done for each dataset; if present, outliers were removed with the ROUT method, and the statistical significance was measured with specific tests as reported in the figure legends. Values * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 were considered statistically significant.

[0163] Results

[0164] The Hspb8_K141N mutation limits cell responsiveness to mTOR-dependent induction Through interaction with the SQSTMl / p62 receptor or the chaperone-assisted selective autophagy (CASA) complex, the HSPB1 and HSPB8 proteins have been shown to be directly engaged in the regulation of the autophagic process (Haidar M et al. Autophagy. 2019:1-18,). When mutated, HSPB1 and HSPB8 significantly impaired the autophagic response; mutant HSPB1 in Hela cells and in motor neurons differentiated from patient-derived induced pluripotent stem cells (iMN) decreased the autophagosome formation (Haidar M et al. Autophagy. 2019:1-18, Tedesco B, et al. Autophagy. 2023;19(8):2217-39). Furthermore, we reported that the Hspb8 knock-in mouse model exhibits progressive accumulation of axoplasmic material with an increase of LC3II and SQSTMl / p62 expression (Bouhy D et al. Acta neuropathologica. 2018;135(l):131-48). It is not yet described whether the most commonly mutated amino acid Lysl41 residue is responsible for a direct deficit in autophagy.

[0165] To verify whether the HPSB8_K141N missense mutation is responsible for defective autophagy in CMT2L, we crossed wild-type Hspb8+ / +or Hspb8K141N / K141Nmice with GFP-LC3tg / tgtransgenic mice and generated mouse lines homozygous for wild-type Hspb8 or for Hspb8K141Nin combination with either one or two copies of the GFP-LC3 transgene. Homozygosity for the Hspb8 K141N mutation was obtained with heterozygosity or homozygosity of the GFP-LC3 transgene. From these mouse lines, embryos were dissected to generate mouse embryonal fibroblasts (MEFs). The generation of the double transgenic Hspb8 / GFP-LC3 lines allowed us to monitor the autophagy activity through quantification of the autophagosome-associated Microtubule-associated proteins 1A / 1B light chain 3B (MAP1LC3B, hereafter referred as LC3), endogenously GFP-tagged, that under autophagy stimulation forms condensed vesicles (hereafter referred as LC3 puncta). The LC3 puncta quantification was used as an estimation of the generated autophagosomes in the murine Hspb8 mutant background.

[0166] After genotypical confirmation, we aimed to test whether the expression of mutant Hspb8 shows a defect in autophagosome formation in the GFP-LC3 MEFs. Autophagy was induced in wild-type or mutant MEFs through canonical mTOR inhibition. Indeed, upon nutrient deprivation, mTOR is inactivated and the autophagy cascade is initiated. Moreover, a combinatorial treatment with 10 nM bafilomycinAl (BafAl) was applied to detect the total autophagy capacity and accumulation of the generated autophagosome. Already with a mild autophagy stimulation by amino acids starvation (50% Earle's balanced salt solution - EBSS), the wild-type Hspb8 cells show a stronger response to autophagy compared to the mutant line. However, the difference became significant when cells were exposed to complete nutrient starvation by serum deprivation. Wild-type cells generated a higher number of autophagosomes under starvation than the mutant cells. However, this effect was exacerbated when the autophagy flux was fully blocked. To confirm that the mutant Hspb8 cells are not able to respond to mTOR-dependent stimulation, wild-type or mutant cells were treated with the canonical mTOR inhibitor rapamycin or with the more potent mTORCl / mTORC2 inhibitor torinl. While wild-type cells responded to pharmacological induction by increasing the number of LC3 puncta per cell, the mutant line showed minimal or almost absent response to the treatment. In addition, the lysosomal inhibitor BafAl leads to a drastic accumulation of LC3 puncta in the wild-type cells, whereas the block of the autophagy flux in the mutant brings a minimal difference. These observations suggest that the MEF expressing mutant Hspb8K141N / K141Ncan only partially sustain the autophagy induction, specifically resulting in mTOR-dependent reduced autophagosome formation.

[0167] In order to exclude that the mutant phenotype was a MEF clonal-specific effect or dependent on the amount of expressed GFP-LC3, multiple Hspb8+ / +or the mutant Hspb8K141N / K141NMEF clones were compared for their capacity to form autophagosomes under rapamycin treatment. The LC3 puncta quantification revealed that at least two unrelated clones, either homozygous or heterozygous for GFP-LC3, showed the same defect suggesting that the K141N mutation is responsible for autophagy defect in MEF, and that canonical mTOR inhibition is not sufficient to induce autophagy, as in the wildtype background. In addition, we confirmed in HeLa cells that overexpression of CMT2L-associated mutations K141E or K141N in human HSPB8 caused a reduction in the number of LC3 puncta compared to the naive Hela cells or cells overexpressing wild-type HSPB8 . These results corroborate the observation that mutant HSPB8 impairs the formation of LC3-positive vesicles under physiological or pharmacological mTOR inhibition, in line with the autophagy defect previously observed for the mutant HSPB1 (Haidar M et al. Autophagy. 2019:1-18).

[0168] Rapamycin induces mitochondrial stress and limits the neuronal growth in iPSC-derived motor neurons

[0169] Autophagosome formation, and consequently autophagy-mediated degradation is compromised by mutations in HSPB1 or HSPB8, pointing to autophagy as a common pathological process in respectively CMT2F and CMT2L. Mutations in HSPB1 and HSPB8 predominantly affect the peripheral nervous system with length-dependent neurodegeneration causing axonal CMT neuropathy. Therefore, we aim to see if routinely used autophagy inducers increase autophagy activation, are well tolerated in a relevant disease cellular model, and might be employed as a CMT therapeutic intervention. iPSC- derived motor neurons carrying the HSPB1_P182L or HSPB8_K141N mutations were exposed to 0.13 pM rapamycin during the entire differentiation (chronic treatment - Rap 0.13 pM) or, after complete maturation at Day 30, for 3 h in combination with BafAl 10 nM (acute treatment - RB 3h). As a marker of autophagy induction, the LC3 cytosolic isoform (LC3I) is processed and lipidated (LC3II) to be incorporated into the growing autophagosome membrane. Upon fusion with the lysosome, LC3II and the cargo, containing the autophagy receptor SQSTMl / p62, are degraded. The chronic treatment with rapamycin revealed LC3II and SQSTMl / p62 degradation, while the exposure to Rap / BafAl led to LC3II and SQSTMl / p62 accumulation (data not shown). However, the 0.13 pM rapamycin treatment caused drastic downregulation of the pre-autophagosome marker ATG9A, and had a limited effect on the formation of LC3 puncta in the iMNs, as reported by the neuronal LC3 intensity that was only partially increased in the mutant and control lines (data not shown). Neurite network deficits and mitochondrial dysfunctions are common traits of CMT-associated genes and other neurodegenerative diseases of the PNS (Van Lent et al. Brain. 2021;144(8):2471-85). Therefore, neurite network shrinkage, neuronal blebbing, and mitochondrial fragmentation are widely used elements to monitor the effects of drug treatments in neurons. Under stress conditions, the mitochondrial network is prone to fragmentation resulting in reduced mitochondrial aspect ratio, reduced size, and increased circularity. Interestingly, the mitochondrial analysis in the iMNs treated with 0.13 pM rapamycin revealed a significant reduction of the aspect ratio both for healthy control and mutant lines . Next, we evaluated the morphological aspects of the neuronal network both in the healthy control and patient-derived lines. The HSPB1 and HSPB8 mutant iMNs were more prone to membrane blebbing compared to the healthy controls, with the HSPB1_P182L line more significantly affected compared to the HSPB8_K141N line, both in number and area of the membrane blebbing. The rapamycin treatment further worsens this phenomenon for all the lines. Strikingly, rapamycin was deleterious for the neurite network development. The growth curve showed that the chronic treatment with 0.13 pM rapamycin profoundly limited the neuronal growth compared to the untreated neurons. Even after the termination of the treatment on Day 30, the network could not recover from the growth inhibition previously received. Similarly, acute exposure to Rap / BafAl, after complete maturation of the neurite network, drastically caused neuronal degeneration. Despite inducing the formation of LC3 puncta and promoting the LC3II and SQSTMl / p62 degradation upon autophagy induction, analysis of the neuronal morphology indicated that rapamycin was neurotoxic and promoted neurodegeneration. Therefore, we aim to identify novel molecules that are surely well tolerated by motor neurons, and, at the same time, could promote the recovery of autophagic deficiencies caused by HSPB1 or HSPB8 mutations.

[0170] Identification of known autophagy inducers to rescue autophagy deficits in Hspb8K141N / K141NMEFs

[0171] As already shown for mutations in HSPB1, we have demonstrated that the HSPB8 missense K141N mutation is responsible for defects in autophagosome formation upon mTOR inhibition with rapamycin. Additionally, the use of rapamycin was neurotoxic for iMN. Therefore, we aim to identify small molecules that might rescue the autophagy deficit reported for mutant HSPB1 or HSPB8 by (i) stimulating the pathway in an mTOR-independent way, or (ii) preparing the cells to be more responsive to mTOR-dependent induction, bringing the LC3 expression up to the wild-type level. Importantly, the novel molecules should be translatable for peripheral neuropathies applications. To understand the contribution in the LC3 puncta formation of the tested molecules in the screening, MEFs were exposed for 22 h to 1 pM of the drug library compounds, followed by a 2 h incubation with the canonical autophagy modulator rapamycin (200 nM) and BafAl (10 nM), after which LC3 puncta formation was quantified. Hspb8K141N / K141NRap / BafAl-treated cells were used as a negative control for the screening to define the canonical basal autophagy response. To identify a positive control (Autophagy Positive control - AP#) and to evaluate the cell responsiveness to the autophagy stimulation, a pilot study was performed by screening a library with known autophagy inducers and inhibitors. The LC3 puncta quantification revealed three hits that were able to stimulate autophagy in the MEF Hspb8 mutant background at least 1,2-fold more than in the wild-type line. The doseresponse curve of the top 3 compounds revealed that all the molecules preserved the cell viability, only Z36 (API) showed cytotoxicity at concentrations higher than 2 pM. SB-216763 (AP3) had a dosedependent increment in the average green intensity which in turn resulted in a higher number of segmented LC3 puncta. Such a drastic fluorescent intensity increment was considered an artifact, therefore, SB-216763 (AP3) was excluded for further analysis. On the other hand, Z36 (API) was chosen as positive control because it showed higher potency ( IC5o below 1 pM and maximal activity at 2 pM) compared to Go6850 (AP2). The two selected positive controls act with two different mechanisms of action. Z36 was discovered as a potent Bcl-XL / Bcl2 inhibitor that specifically can disrupt the interaction between Bcl2 and Beclinl, thus promoting autophagy induction. Go6850 inhibits members of the protein kinase C (PKC) family, which have a role in the autophagy activity downregulation. It is worth noting that the in vitro assay design is intended to particularly identify molecules that enhance autophagy induction. For instance, the pretreatment with the autophagy inhibitor bafilomycin Al did not result in an increase in the number of LC3 puncta compared to the control. Next, Z36 was used to determine the robustness and quality of the screening. With a Z' factor of 0,64 and a coefficient of variation (CV) of 0.11, the assay was evaluated optimal to proceed with the primary screening.

[0172] Identification of novel autophagy enhancer in Hspb8K141N / K141NMEFs

[0173] A phenotypic drug screening was conducted to identify novel autophagy enhancers that could rescue the autophagic defects in the presence of mutant HSPB8. The Microsource Spectrum Collection library contains FDA / EMA-approved molecules, annotated compounds and natural products that are currently in use or have been used for different clinical implications. The library was chosen for its pharmacological and chemical diversity, thus increasing the probability of identifying and repositioning one of these molecules as autophagy inducers for the HSPBs mutant models. The primary screening was based on the GFP-LC3 puncta quantification in Hspb8K141N / K141NMEF cells. The compound Z36 (1 pM) and DMSO were used as positive and negative controls respectively (Fig. la). The primary hits were validated through a dose-response curve to evaluate potency and toxicity. We made use of the knowledge about the annotated compounds in PubChem and ChEMBL Database, as well as literature-based investigation of the translational potential of each compound to select the hits to be further validated on MEFs by immunoblotting and in human iPSC-derived motor neurons (iMNs).

[0174] The Hspb8K141N / K141NMEF cells were exposed for 22 h to the library compounds, followed by the canonical autophagy induction with Rap / BafAl. The number of GFP-LC3 puncta was quantified per cell and reported as percentage to control (POC - DMSO-treated cells) (Fig. la). The quality of the screening results was assessed by calculating the CV and Z' factor, and the correlation between replicates for the GFP-LC3 puncta count. The Pearson correlation coefficient revealed high consistency between replicates (r = 0,99). Cell number and green fluorescence intensity were used as primary parameters to filter the entire compound list. In details, only the molecules that satisfy the following imposed filters in both technical replicates were considered positive hits: (i) compounds that did not reduce the cell count more that 50% compared to the DMSO [cell count (POC) >50%], (ii) compounds with an increment of the fluorescence intensity <200% compared to the DMSO [average green intensity (POC)<200%] , and (iii) compounds with an increment of GFP-LC3 puncta per cell >120% to the DMSO [LC3 puncta count (POC) >120%]. However, from the first set of 24 hits, 8 molecules were excluded based on the chemical reactivity, drug-likeness, mechanism of action, and previous clinical applications (Fig. lb). Furthermore, the remaining 16 hits were divided into 2 groups (Fig. 1, and Table 1 below.

[0175] Table 1

[0176] CellLC3count puncta Name Cas number Bioactivity Source count (POC) (poc)

[0177] Hit group A: LC3 puncta count (POC) > 300%

[0178] Merbromin 91,57 5074,24 129-16-8 antibacterial synthetic Rutilantinone - Streptomvces

[0179] 89,74 2947,41 21288-61-9 coccidiostat

[0180] MCI spp

[0181] 7232-51-1,

[0182] Pararosaniline 569-61-9 anthelmintic,

[0183] 96,84 1646,30 synthetic pamoate - MC2 [pararosanilinantischistosoma| e]

[0184] Aklavine

[0185] 60504-57-6 antibacterial, Actinomyces hydrochloride - 82,01 1305,87

[0186] [aklavine] antineoplastic spp

[0187] 6151-30-0,

[0188] 69 05 6 anthelmintic,

[0189] Quinacrine Antimalarial

[0190] 91,01 918,28 [anhydrous], antimaianai, synthetic hydrochloride

[0191] 83-89-6 • int .erca |lat ..ing agent

[0192] [quinacrine]

[0193] Bisanhydrorutilan Streptomyces

[0194] 91,41 572,05 749-18-8 antibacterial tinone - MC4 spp

[0195] Pyrvinium

[0196] 58,69 416,87 3546-41-6 anthelmintic synthetic pamoate

[0197] Hit group B: LC3 puncta count (POC) 120% - 300%

[0198] Ergocalciferol - irradiation of

[0199] 97,31 218,49 50-14-6 antirachitic vitamin

[0200] MC5 ergosterol

[0201] 1,4- coal tar

[0202] 83,91 181,55 130-15-4 NaN

[0203] Naphthoquinone product antineoplastic, anti-

[0204] Celastrus inflammatory, NO scandens &

[0205] Celastrol - MC6 58,69 175,58 34157-83-0 synthesis inhibitor,

[0206] Tripterygium chaperone wilfordii stimulant

[0207] Kinetin Riboside 80,29 168,61 NaN NaN semisynthetic

[0208] Desoxycorticoster

[0209] 85,60 158,38 56-47-3 mineralocorticoid adrenocortex on acetate antimethemoglobin

[0210] Methylene blue 95,47 157,37 7220-79-3emic' synthetic cyanide antidote anti-asthma, Piplartine - MC7 60,24 153,03 20069-09-4 Piper spp antibronchitis

[0211] Artemisia

[0212] Artemisinin - MC8 91,11 148,99 63968-64-9 antimalarial annua

[0213] 55812-90-3,

[0214] Dexamethasone glucocorticoid, anti-

[0215] 92,91 142,20 1177-87-3 semisynthetic acetate inflammatory

[0216] [anhydrous]

[0217] Z36 - AP1 71,15 1798,00

[0218] DMSO 99,41 104,40

[0219] List of the top compounds selected from the primary screening. The Hspb8K141N / K141N MEFs clones were exposed to the compounds in the Spectrum Collection library (Microsource Discovery Systems). The LC3 puncta per cell were quantified and compounds were ranked based on the LC3 puncta count (POC). The hits were divided into two groups depending on their potency. Hit group A of potent inducers [LC3 puncta count (POC) > 300%] and Hit group B of milder inducers [LC3 puncta count (POC): 120% - 300%].

[0220] Hit group A presents a GFP-LC3 puncta 3-fold change compared to the DMSO-treated cells [LC3 puncta count (POC) > 300%], it includes strong inducers that lead to an accumulation of autophagosome in the cells, up to 50 folds more than DMSO. This is the case of the first hit, merbromin, a known organic mercury compound used as an antiseptic agent (https: / / go.drugbank.com / drugs / DB13392). However, merbromin is a fluorescein and the use of merbromin has been dismissed in many countries due to possible poisoning. Therefore, this hit was excluded in the next validation for its limited translational potential in neuropathic treatment. A second set of molecules, Hit group B, was identified as milder inducers that generate a GFP-LC3 puncta increment of about 0.5-fold more than DMSO-treated cells [LC3 puncta count (POC): 150% - 300%]. This categorization allows us to evaluate the effect of molecules with a larger variety of chemical properties, mechanism of action, and potency on the autophagy induction.

[0221] As further validation of the robustness of the primary screening, we inspected the chemical space of the library and checked whether the hit selected in the primary screening shared chemical similarities. The most representative components were chosen to generate a reduced 2D-space in which each molecule acquires a position in the space. Isolated and distant points indicate highly diverse molecules whereas molecules with high similarity cluster together. The PCA analysis revealed two main clusters that accommodate 10 hits in total, suggesting that our assay highlighted small molecules with chemical similarity, and share the ability to induce autophagy in MEFs (Fig. 4). Taking into account the chemical and biological activity, the primary screening successfully provided a set of potent and mild autophagy enhancers for further validation.

[0222] Validation of selected compounds that induce autophagy in the Hspb8K141N / K141NMEFs

[0223] To confirm the drug selectivity for the mutant Hspb8 line and evaluate their potency, a dose-response curve was performed on the 16 hits to validate the GFP-LC3 puncta formation in a concentration range from 0,25 pM to 8 pM, both on the Hspb8+ / +and the Hspb8K141N / K141NMEF clones. In parallel, cell number was assessed in a dose-dependent manner to determine the cellular tolerance to the drug treatment (Fig. 5a). Only compounds that preserved cell viability and overall fluorescence intensity were considered for further validation. The analysis revealed that eight molecules significantly increased the GFP-LC3 puncta in a dose-dependent manner with the maximal effect higher in the mutant Hspb8 line compared to the wild-type line. To confirm the autophagy stimulation upon treatment with the hits, we monitored changes in the autophagy-specific markers LC3, SQSTMl / p62 and ATG9A through western blot, specifically if the exposure with the selected drugs caused LC3 species or SQSTM1 degradation or accumulation. Thus, Hspb8K141N / K141NMEFs were pre-treated for 22 h with each hit compound (1 pM), along with the screening positive controls (1 pM Z36 or Go6850, respectively API and AP2), BafAl (10 nM), chosen as reference autophagy inhibitor, and in combination with 2 h stimulation with rapamycin (200 nM) and bafilomycin (10 nM).

[0224] The validation of the first set of hits indicated that MC2 treatment stimulated LC3II breakdown, by preserving the lysosomal degradation. Moreover, upon combination with rapamycin, MC2 is able to enhance the autophagy response more effectively than other compounds in group A (Fig. 5b).

[0225] When MC5 and MC7 were combined with the rapamycin stimulation, the LC3II / LC3I ratio increased similarly to the positive control AP2, suggesting that these small molecules are able to sustain and enhance the LC3 ratio under canonical induction (Fig. 5c). The LC3 / SQSTM1 immunoblotting revealed that MC5 and MC7 preserved the SQSTM1 degradation and promoted a mild increase in the LC3II / LC3I ratio, in line with the result of the primary screening.

[0226] To determine whether autophagy is preserved and stimulated already in the early stage of the pathway, we monitored the expression of ATG9A, the only other transmembrane protein, besides LC3, in the entire process Processed forms of ATG9A were abundantly detected and increased specifically with MC2 and MC4 (data not shown and Fig. 5d). Cleaved ATG9A forms were also enriched upon treatment with MC7 and MC8 (data not shown and Fig. 5e). From 7 compounds validated from the primary screening, pararosaniline pamoate (MC2) and piplartine (MC7) were considered for further validation, based on their ability to promote autophagosomes formation with high specificity for the mutant Hspb8 background, promote the LC3II degradation, preserve the ATG9A and SQSTMl / p62 expression during the autophagy stimulation, and more importantly, enhance the autophagy response of the Hspb8 mutant cells under rapamycin treatment.

[0227] Pararosaniline pamoate and piplartine preserve the autophagic flux

[0228] Autophagy is a dynamic process that sees the formation of autophagosomes after the initial induction, and its degradation upon fusion with the lysosomes. To determine the extent of the degradation through the lysosomes and evaluate how many autophagosomes are formed during the drug treatment, we made use of a combinatorial treatment with bafilomycin Al (BafAl). BafAl is a lysosomal inhibitor that blocks the lysosomal degradation, thus ensuring the accumulation of the autophagic vesicles in the cytosol. The difference between cells treated with or without bafilomycin provides an estimation of the autophagic flux. Pararosaniline pamoate (MC2) and piplartine (MC7) were tested along with DMSO and the two positive controls, Z36 (API) and Go6850 (AP2), for a 4-hour time window, to capture the effect of the drug in the early stage of the induction, without causing cytotoxicity due to the BafAl exposure. The same samples were processed for western blotting (WB) and transmission electron microscopy (TEM). The LC3 and SQSTM1 immunoblotting confirmed that API was a potent inducer due to the increment in LC3II (Fig. 5f). However, the differences between API-treated cells and those exposed to APl / BafAl is reduced compared to the DM SO-treated cells, suggesting that API might cause a reduction in the autophagy flux (Fig. 5g). A 4 h treatment with MC2 and MC7 promoted a mild reduction in LC3II, compared to DMSO-treated cells, which accumulated only when MC2 or MC7 were combined with BafAl (Fig. 5f-g). The TEM imaging revealed that MC2 and MC7-treted cells were enriched in autophagic vacuoles, spanning from autophagosomes to autolysosomes at different stages of the pathway (Fig. 5h). Based on these observations, we could confirm that the exposure to MC2 and MC7 promoted the formation of autophagic vacuoles while preserving the continuous autophagy flux. Moreover, after the evaluation of the limited cytotoxicity, pararosaniline pamoate (MC2) and piplartine (MC7) were chosen as the best candidate hits to be tested on iPSC-derived motor neurons.

[0229] The novel-identified molecules induce autophagy in iMNs

[0230] We have previously reported that CMT-associated mutations in HSPB1 and HSPB8 result in a partial reduction in the neurite network in iPSC-derived motor neurons (iMNs) along with mitochondria defects (Van Lent et al., above). In this study, we could confirm that already at Day 30 the HSPB1_P182L iMNs presents a significantly reduced neuronal network area compared to the two healthy controls, while the HSPB8_K141N showed similarity to the control lines. Furthermore, we monitored the neuronal blebbing, alteration of the neuronal membrane that occurs in case of stress, neuronal damage, and ultimately apoptosis. The assessment of neuronal blebbing in untreated iMNs revealed that the HSPB1_P182L iMNs were considerably more affected by neuronal stress compared to the control lines, similarly, the HSPB8_K141N mutant line was more prone to axonal degeneration. To test whether the two hits have any potential to induce autophagy and ameliorate the neuronal phenotype in the relevant CMT neuronal cellular model, iMNs carrying the HSPB1_P182L or HSPB1_K141N mutation were exposed during the differentiation to pararosaniline pamoate (MC2) or piplartine (MC7). Next autophagy induction was quantified, followed by the neuronal phenotype analysis.

[0231] Importantly, to avoid the drug treatment contributing to a stress response in the iMN and jeopardizing the autophagy stimulation, the optimal drug concentration for the iMN was determined using a doseresponse curve (DRC) in a range of 0.125 to 2 pM for the two positive controls (API and AP2) and the two hit compounds (MC2 and MC7). The neurite network was monitored over the entire period of the differentiation through quantification of the thick neurite bundles and thinner neurites (Fig. 6). To test the possible drug treatment-induced neurotoxicity, the DRC was performed on iMN derived from a single healthy control iPSC line and the NEFL_P8R line (an iPSC line carrying a Pro8Arg missense mutation in Neurofilament Light protein associated with CMT2E), which was chosen as the most sensitive line to neurotoxicity due to its severe neuronal defect. Starting from Day 18, after only 4 days from the beginning of the drug treatment, the motor neurons had a significant loss in the neurite network when treated with the highest concentration of API, MC2 and MC7 (Fig. 6a-c-d). Only AP2 was better tolerated (Fig. 6b). However, at the end of the differentiation (Day 36) the concentrations that promoted a better development of the neurite network were in the lower range of the curve (Fig. 6e). Therefore 0.13 pM / 0.25 pM API, MC2 and MC7, 0.25 pM / 0.5 pM AP2 were considered the optimal concentrations for the iMNs and applied on the HSPB1_P182L and HSPB8_K141N iMN differentiation, as shown in the experimental plan (Fig. 2a).

[0232] Previous structural studies on the HSPBs have shown that the C-terminal mutation P182L in HSPB1 forces the chaperone to form large oligomers due to the opening of a protein cleft that traps the chaperone itself and other cytosolic proteins. As a consequence of this, we observed that in the HSPB1_P182L iMN, SQSTMl / p62 accumulates in the insoluble fraction (Fig. 2b right). MC2 promoted a mild reduction in the LC3II / LC3I ratio along with the reduction in SQSTMl / p62 both in the soluble fraction and insoluble fraction, while preserving the pre-autophagosome marker expression ATG9A (Fig. 2b left). These elements together indicated that the autophagy flux was not perturbed. Moreover, MC2 promotes a dose-dependent increment in the neuronal LC3 intensity of the segmented autophagic vesicles in the HSPB1_P182L iMN (Fig. 2c). Similarly, in the HSPB8_K141N iMN, MC2 reports a milder increment in the LC3 neuronal intensity. However, the immunoblotting shows a clear reduction of p62 / SQSTMl compared to the untreated line (Fig. 2b). MC7 (piplartine) was selected as the best compound from the set of milder autophagy enhancers (Hit group B) and tested on the mutant HSPB iMN lines in the same experimental plan as MC2. In both mutant lines, MC7 increased the LC3II expression with a reduction in the p62 / SQSTMl expression in the soluble fraction. However, in the insoluble fraction p62 was enriched, probably because SQSTM1 got condensed in p62 bodies (Fig. 2b right). MC7 treatment causes a dose-dependent increase in the neuronal LC3 intensity in both HSPB1 and HSPB8 iMN lines with a pronounced effect on the HSPB1 line (Fig. 2c). The validation on iMN demonstrated that MC2 and MC7 treatment promoted autophagy resulting in an increase in the LC3-positive vesicles in the iMN and relocation of the autophagy receptor SQSTMl / p62 (Fig. 2).

[0233] Pararosaniline pamoate and piplartine ameliorate the neurite network in iMN

[0234] Next, we aimed to determine whether the newly discovered autophagy inducers (pararosaniline pamoate = MC2 and piplartine = MC7) affected the overall neuronal phenotype, including alteration of the neuronal membrane, mitochondrial morphology, and neurite outgrowth. These aspects are crucial not only for monitoring the treatment tolerance, but also to evaluate the amelioration of the neuronal defects that are directly associated with the expression of the CMT mutations in HSPB1 or HSPB8., We have shown that the HSPB1_P182L iMN developed a significantly less dense network compared to the healthy controls and numerous blebbing points along the neurites, as a clear sign of neuronal stress. Despite the HSPB8_K141N iMN not yet manifesting a significant defect in the overall neurite network, alterations of the neuronal membranes could be observed (this can foresee a defect in the neurite network in later stages of the motor neuron maturation). Upon treatment with MC2, we observed a reduction of the neuronal blebbing in a dose-dependent manner in the HSPB1_P182L line (Fig. 3a-b). For the HSPB8_K141N iMN, the neuronal blebbing was unaltered since the untreated HSPB8 line already showed a similar phenotype compared to the healthy control lines (Fig. 3a-c). Another typical sign of neuronal stress is mitochondrial fragmentation, a phenotype commonly associated with severe mutations in axonal CMT and other neurodegenerative diseases. Indeed, we have shown that the autophagy induction mediated by rapamycin resulted in a significant reduction of the mitochondrial aspect ratio in both control and patient-derived motor neurons. On the contrary, the MC2 treatment in the HSPB1_P182L iMN generated a partial increment of the mitochondrial aspect ratio while in the HSPB8 mutant line, a significant improvement of the mitochondrial morphology was observed (reported as an increase in the mitochondrial aspect ratio) (Fig. 3e). Despite the amelioration at the neuronal level in the iMN expressing the HSPB1_P182L mutation, MC2 reported only a milder increment of the growth speed, keeping the total neurite area still unaltered (Fig. 3f). On the other hand, MC2 significantly increased the neurite area of the HSPB8_K141N line with a higher growth speed compared to DM SO-treated cells (Fig. 3g).

[0235] The second molecule validated on the iMN, piplartine (MC7), significantly reduced the presence of neuronal blebbing in the HSPB1_P182L line, while it did not affect the neuronal morphology in the HSPB8_K141N line (Fig. 3b-c). Under MC7 treatment, the mitochondrial aspect ratio was not significantly reduced in the HSPB1_P182L iMNs, as well as in the HSPB8_K141N at the lowest concentration, suggesting that MC7 was tolerated by the neurons (Fig. 3e). Strikingly, MC7 treatment induced an amelioration of the neurite network during the entire differentiation with an increment of both the total area and growth speed for the HSPB1_P182L iMN, and more significantly for the HSPB8_K141N mutant line (Fig. 3 f-g). The positive effect exerted on the mutant HSPB1 and HSPB8 motor neurons pinpoints MC7 as an optimal candidate to be further tested in preclinical studies for CMT2F and CMT2L (Fig. 3h).

[0236] Effects on AKT phosphorylation

[0237] Transcriptomic analysis on CMT2 iMN revealed alteration in the PI3K-Akt signaling. Specifically, CMT patient-derived lines showed an increase in AKT phosphorylation (Ser473) compared to control lines. Therefore, it was tested whether MC2 or MC7 treatment could reestablish the AKT phosphorylation level comparable to controls, as well as other transcriptional regulators (KHDRBS2 and NKX6.1) involved in motor neuron differentiation. The p(Ser473)AKT:AKT ratio confirmed that HSPB1_P182L and HSPB8_K141N presented a higher phosphorylation level than the controls. Interestingly, MC2 and MC7 reduced the AKT phosphorylation, with a major effect on the HSPB1_P182L line. The rescue of the AKT activation status, together with the amelioration of the neuronal phenotypes on the mutant HSPB1 and HSPB8 motor neurons, pinpoints MC7 as an optimal candidate to be further tested in preclinical studies for CMT2F and CMT2L. AKT is a connecting point between the metabolic stress response and autophagy regulation. AKT can be activated in case of ER stress or by the metabolic sensor mTORC2. The reduction in p(Ser473) AKT phosphorylation by pararosaniline pamoate or piplartine indicates an inhibition of mTORC2 activation and mTORC2 / Akt-dependent mechanism.

Claims

CLAIMS1. A therapeutic agent for use in the treatment and / or prevention of a neuropathy, myopathy or cardiopathy in a subject in need thereof, wherein said subject is characterized by the presence of one or more mutations in a gene associated with autophagy in said subject; and wherein said therapeutic agent is piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof.

2. The therapeutic agent for use according to claim 1, wherein said one or more mutations are one or more mutations in the Heat Shock Protein Family B Member 1 (HSPB1) gene, Heat Shock Protein Family B Member 3 (HSPB3) gene, Heat Shock Protein Family B Member 5 (HSPB5) gene, Heat Shock Protein Family B Member 6 (HSPB6) gene, Heat Shock Protein Family B Member 8 (HSPB8) gene, neurofilament light chain (NEFL) gene, Ras-related protein Rab-7a (RAB7a) gene, Leucine Rich Repeat And Sterile Alpha Motif Containing 1 (LRSAM1) gene, Tripartite Motif Containing 2 (TRIM2) gene, Beclin-1 (BECN1) gene, Transient Receptor Potential Cation Channel Subfamily V Member 4 (TRVP4) gene, Mitofusin-2 (MFN2) gene, Dynamin-2 (DNM2) gene, DnaJ heat shock protein family (Hsp40) member B2 (DNAJB2) gene, Member B of the family with sequence similarity 134 (FAM134B) gene, Peripheral myelin protein 22 (PMP22) gene, BAG family molecular chaperone regulator 3 (BAG3) gene, transient receptor potential cation channel subfamily V member 4 (TRPV4) gene, VAMP associated protein B (VAPB) gene, WNK lysine deficient protein kinase 1 (WNK1) gene, seipin lipid droplet biogenesis associated (Seipin) gene, ganglioside induced differentiation associated protein 1 (GDAP1) gene, N-myc downstream regulated 1 (NDRG1) gene, tectonin beta-propeller repeat containing 2 (TECPR2) gene, lipopolysaccharide induced TNF factor (LITAF) gene, SH3 domain and tetratricopeptide repeats 2 (SH3TC2) gene, kinesin family member 1A (KIF1A) gene, tyrosine kinase A (TrkA) gene, dynactin 1 (DCTN1) gene, dystonin (DST) gene, valosin containing protein (VCP) gene, phosphoinositide 5-phosphatase (FIG4) gene, myotubularin-related protein 2 (MTMR2) gene, myotubularin-related protein 13 (MTMR13) gene, or chaperonin containing TCP1 subunit 5 (CCT5) gene.

3. The therapeutic agent for use according to claim 1, wherein said one or more mutations are one or more mutations of Table A.

4. The therapeutic agent for use according to any of claims 1 to 3, wherein said one or more mutations are one or more mutations in the Heat Shock Protein Family B Member 1 (HSPB1), Heat Shock Protein Family B Member 8 (HSPB8) and / or neurofilament light chain gene (NEFL).

5. The therapeutic agent for use according to any of claims 1 to 4, wherein said one or more mutations comprise a mutation which ensures an amino acid change in the alpha crystallin domain of the HSPB1 protein.

6. The therapeutic agent for use according to any of claims 1 to 5, wherein said one or more mutations comprise a mutation which ensures an amino acid change at K141 in the HSPB8 protein.

7. The therapeutic agent for use according to any of claims 1 to 6, wherein said one or more mutations comprise a mutation which ensures an amino acid change in the head domain of the NEFL protein, such as in the P8 or P22 position.

8. The therapeutic agent for use according to any of claims 1 to 7 , wherein said disease or disorder is a hereditary disease.

9. The therapeutic agent for use according to any of claims 1 to 8, wherein said disease or disorder is a peripheral neuropathy, or a distal myopathy.

10. The therapeutic agent for use according to claim 9, wherein said peripheral neuropathy is Charcot- Marie-Tooth (CMT) disease, preferably CMT2 disease.

11. The therapeutic agent for use according to claim 10, wherein said wherein said CMT2 disease is selected from the following subtypes: patients diagnosed with hereditary motor and sensory neuropathy (HMSN); or patients with predominantly motor involvement, known as distal hereditary motor neuropathy (dHMN), axonal CMT or intermediate subtypes.

12. The therapeutic agent for use according to any of claims 1 to 11, wherein said subject in need thereof has been diagnosed with a disease or disorder associated with the presence of one or more mutations in a gene involved in autophagy, preferably the presence of a mutation in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, BAG3, TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5, more preferably the presence of a mutation in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG3, most preferably the presence of a mutation in HSPB1, HSPB8 and / or NEFL.

13. The therapeutic agent for use according to any of claims 1 to 12, wherein said subject in need thereof has been diagnosed with an autophagy deficiency.

14. The therapeutic agent for use according to any of claims 1 to 13, wherein the therapeutic agent is comprised in a pharmacological composition, preferably said pharmacological composition further comprises one or more pharmaceutically acceptable carriers.

15. A method for determining the susceptibility of a subject suffering from an autophagy-related disease or disorder to the treatment with piplartine, pararosaniline pamoate, or an analogue, derivative or prodrug thereof, the method comprising determining whether said subject is characterized by the presence of one or more mutations in HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, BAG3, TRPV4, VAPB, WNK1, Seipin, GDAP1, NDRG1, TECPR2, LITAF, SH3TC2, KIF1A, TrkA, DCTN1, DST, VCP, FIG4, MTMR2, MTMR13, and / or CCT5, preferably HSPB1, HSPB3, HSPB5, HSPB6, HSPB8, NEFL, RAB7a, LRSAM1, TRIM2, BECN1, TRVP4, MFN2, DNM2, DNAJB2, FAM134B, PMP22, and / or BAG3.

16. The method according to claim 15, comprising determining whether said subject is characterized by the presence of one or more mutations of Table A.

17. The method according to claim 15, comprising determining whether said subject is characterized by the presence of one or more mutations in HSPB1, HSPB8 and / or NEFL in its genome.

18. An in vitro method for determining the suitability of an agent for the treatment and / or prevention of an autophagy-related disease or disorder in a subject, comprising the steps of: a) determining whether a test agent is capable of rescuing an autophagy deficiency in a cell; and b) comparing the autophagy-rescuing ability of said test agent as determined in a) to the autophagyrescuing ability of piplartine, pararosaniline pamoate or an analogue, derivative or prodrug thereof in said cell.

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