Lysosomal storage disorders (LSDs) are a heterogenic subgroup of more than 60 rare inborn inherited metabolic disorders (Burton, 1998; Winchester et al., 2000). LSDs were first diagnosed in the 19th century long before lysosomes were even identified in the cell by Christian de Duve in 1955 and therefore were not yet classified as LSDs at that time (de Duve et al., 1955; de Duve, 2005). The LSDs classification evolved subsequent to our improved understanding of the function of lysosomes and the identification of their biogenesis and enzymatic proteins. Lysosomes are specialized sacs housing hydrolytic enzymes to digest various cellular substrates to be recycled and delivered to targeted sites within the cell (Hunziker and Geuze, 1996). Individual LSDs are usually caused by deficiencies in specific lysosomal enzymes due to genetic defects in their coding genes (Grayson, 2016). Genetic defects will either disrupt the expression of the mutated protein or result in the expression of a structurally or functionally defective enzyme. In all cases, the residual enzyme activity reaches a level below the cellular threshold required for normal biological function. The major outcome of low lysosomal enzyme activity is the accumulation of its substrates in lysosomes leading to toxicity, cell swelling, and death (Ballabio and Gieselmann, 2009). This can occur in multiple tissues including muscle, eye, liver, spleen, bones, and joints. However, the most serious consequences arise when the deficient enzyme function is crucial for neuronal cells, which in fact is observed for most LSDs (Begley et al., 2008). A minor group of LSDs such as Mucolipidosis type II/III and Niemann–Pick disease type C1 are caused by defects in non-enzymatic lysosomal proteins, lysosomal membrane proteins and enzymatic co-factors rather than acidic hydrolases (Saftig and Klumperman, 2009).

In this review, we aim to present a general overview of the molecular and cellular bases of LSDs and highlight recent advances on our understanding of proteostasis manipulation as a therapeutic target for some LSDs. A brief description of the normal proteostasis and lysosomal network has been stated with a deeper insight into the biochemical and molecular mechanisms underlying different LSDs. Currently available LSDs treatments aim to reduce the accumulation of substrates in lysosomes. Enzyme replacement therapy (ERT) has been approved long ago for several LSDs but recently small molecular compounds manipulating proteostasis have been introduced in clinics and research laboratories to overcome limitations and inadequacies of the previous therapies. Pharmaceutical chaperones (PCs) are specific small molecular weight compounds that specifically bind and stabilize mutated enzymes while proteostasis regulatory compounds act generally on specific components of the proteostasis pathways to enhance protein folding by increasing cellular proteostasis capacity (Parenti et al., 2013). The focus of this article is to present recent advances on PCs and proteostasis regulatory compounds that were clinically and/or experimentally shown to be promising at correcting the defects and to summarize our understanding of their molecular bases by which they exert their effects.

Lysosomal storage disorders are mainly inherited as autosomal recessive disorders except for Fabry, Hunter, and Danon disorders which are inherited in an X-linked recessive manner (Ballabio and Gieselmann, 2009). The genotype and phenotype correlation of LSDs has been extensively studied to link symptoms to the genetic defect but little is known so far in understanding the cellular mechanisms by which the mutation causes the underlying disorder. Although disease symptoms are more linked to the type of the accumulated substrate and its location, however, LSDs pathogenic effects on lysosomal enzymes processes and pathways are not fully understood yet. All types of mutations were detected in LSDs with various effects resulting in heterogeneity of symptoms and severity. The most severe form of LSDs is the complete loss of a lysosomal enzyme due to protein truncations as seen with some indels (causing frameshifts) and non-sense mutations. In addition, for some LSDs, functional mRNA generation is blocked due to splice site mutations resulting in almost complete loss of the enzyme or very low residual activity when a small amount of the transcript is normally processed and translated (McInnes et al., 1992). Missense mutations are very frequent in LSDs but their effects are the most difficult to establish especially their cellular mechanisms. Missense mutations effects depend mostly on the site of change at the protein level. Amino acid substitution changes in the enzyme active site are believed to be the most deleterious leading to almost complete loss of residual enzyme activity (Zhang et al., 2000; Garman and Garboczi, 2004; Qian et al., 2015). Missense mutations occurring outside the active site may affect the folding properties and trafficking of the mutated protein, hence, its possible retention in the endoplasmic reticulum (ER) by the ER quality control machinery. ER retention leads to the complete loss of enzyme activity due to mislocalization of the lysosomal enzyme or premature degradation through the proteasomal degradation by endoplasmic-reticulum-associated protein degradation (ERAD) (Ron and Horowitz, 2005; Wang et al., 2011b). In both cases, the mutated enzyme does not successfully reach lysosomes to perform its function. In such cases, small molecular chaperones have been proposed and tested as potential therapies to correct the effect of such structural mutations (Khanna et al., 2014; Hossain et al., 2015; Laigre et al., 2016).

Lysosomal storage disorders are clinically heterogeneous with wide range of symptoms even within the same disorder. The main hallmark among all LSDs is the accumulation of metabolic substrates within lysosomes leading to cell dysfunction and eventually cell death. Accumulation of undigested metabolites results in the activation of several cellular pathogenesis pathways leading to multi-systematic clinical manifestations (Futerman and van Meer, 2004). LSDs are progressive with most of the patients born healthy with no signs of the disease. Symptoms start appearing within a period of time depending on the underlying mutation, affected tissue and the biochemistry of the accumulated substrate(s) (Platt et al., 2012). LSDs are classified based on the type of the accumulated macromolecules into glycan, lipid, and protein degradation defects in addition to subgroups with affected trafficking and lysosomal protein transporters (Murthy et al., 2010). Most of LSDs belong to the glycan defected subgroup harboring about 30 different disorders. Gangliosides, galactosylceramide, and sulfatide serve important functions in the brain tissue and patients with LSDs (as seen in GM1- and GM2-gangliosidosis) who show cellular accumulation of the mentioned substrates suffer from variant CNS complications ranging from seizures to intellectual disabilities (Sandhoff and Harzer, 2013). The severity of neurological presentation correlate with the site of accumulated substrates which can be the cortex, thalamus, cerebellum, and hippocampus (Platt et al., 2012). On the other hand, some of the LSDs do not have central nervous system (CNS) involvement. Recently, LSDs has been classified according to the defective protein as the previous classification can be misleading for some disorders (Wraith, 2011). For some LSDs more than one substrate is accumulated in tissues as seen in GM1-gangliosidoses patients who show defects in oligosaccharides, sphingolipids, and keratan sulfate degradation collectively (Sandhoff and Harzer, 2013). Mucolipidosis disorders on the other hand show glycoproteins and gangliosides aggregates rather than mucolipids as proposed by its name (Paik et al., 2005). Based on the disease age of onset, multiple LSDs like GM1-gangliosidosis are classified into infantile, juvenile, and adult forms where the classical LSD is the infantile form which is mostly associated with CNS manifestations leading to poor life expectancy of the affected child (Wraith, 2002). Symptoms might appear as early as in uterus or directly after delivery whereas in some cases the child is born healthy and symptoms appear after few months and progress, often rapidly. Adulthood form of LSDs usually have milder manifestation of the disease and patients tend to have better life expectancy (Platt et al., 2012).

In addition to the rarity of LSDs incidence, clinical phenotype overlapping between disorders as well as the delay or misdiagnoses of many disorders contribute to the low amount of data reported and the poor epidemiological coverage of LSDs. Individual LSDs are rare worldwide but collectively they are relatively common with a prevalence of ∼1/5175 live births (Sanderson et al., 2006). High frequency was found in an Emirati study conducted in 2013 with a total incidence of 27 per 100,000 live births which was close to the data obtained from a Portuguese study in 2004 (Pinto et al., 2004; Al-Jasmi et al., 2013). Gaucher is the most common type of LSDs worldwide with a ∼1/75,000 live births followed by Fabry disease with a 1/100,000 live births incidence (Grabowski, 2005; Germain, 2010). LSDs incidence on the other hand is relatively high in genetically isolated communities such as the Ashkenazi Jews who showed a prevalence as high as 1 in 855 live births for Gaucher’s disease (Vallance and Ford, 2003).

To understand the molecular and cellular mechanisms behind LSDs, we need to understand the concept of proteostasis and lysosomal dynamics. Proteostasis is the combination of multiple regulatory integrated systems including protein synthesis, structural folding, post-translational modification, trafficking and degradation (Labbadia and Morimoto, 2015). Lysosomal enzymes are glycoproteins synthesized in the ER with a specific N-terminal signal sequence. Several glycosylation events occur in the ER to enhance protein folding with the help of several ER-resident enzymes and molecular chaperones. Protein glycosylation and loss of the N-terminal signal initiate enzyme folding and translocation to the Golgi. Once in the Golgi, most lysosomal enzymes undergo a phosphotransferase and diesterase enzymatic processes to acquire a mannose 6-phosphate (M6-P) moiety that is important for protein translocation into lysosomes via M6-P receptors expressed on lysosomal membranes (Coutinho et al., 2012). The protein-receptor complex dissociates under lysosomal acidic environment where receptors are recycled back to Golgi for another round leaving the enzyme within lysosomes. Lysosomal acidity activates the trafficked enzyme through multiple proteolytic and/or folding processes to achieve the fully functional active enzyme conformation (Vellodi, 2005).

Proteins that fail to fold properly in ER, may aggregate disrupting cellular haemostasis in a condition known as ER stress (ERS). A cell under ERS activates various signaling pathways and processes including unfolded protein response (UPR) to restore its normal state through the expression of various genes functioning as chaperones to enhance protein folding, translational inhibitors to stop protein flux into ER, and activators of ERAD machinery (Xu et al., 2005). In the case of chronic UPR when a cell fails to reach haemostasis, specific apoptotic pathways are activated leading to cell death. UPR gene expression initiates the activation of three signaling pathways in the ER membrane; inositol-requiring protein 1 (IRE1), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) (Shen et al., 2004; Xu et al., 2005). The activation of IRE1 pathway by self-oligomerize and phosphorylation initiates the expression of the ERAD components. The PERK pathway on the other hand inhibits mRNA translation resulting in less protein flux to ER. ATF6 is an ER transmembrane transcription factor that is transported to Golgi where its cytosolic peptide gets cleaved by the site-2 protease (S2P) to be translocated to the nucleus and activates the transcription of ER protein folding chaperones (Tsai and Weissman, 2010; Walter and Ron, 2011).

As part of the UPR system, degradation of misfolded proteins that failed to fold properly is carried out by the ERAD machinery. ERAD is a highly orchestrated protein machinery that function in the cell’s ER and cytosol in four main steps. The process starts with recognizing misfolded proteins via highly specific chaperones through motifs like hydrophobic patches, N-glycan moieties, and disulphide bonds. Then substrates are targeted to retrotranslocation and ubiquitination. Retrotranslocation is an ATP-dependant step at which targeted proteins are translocated to the cytosol via the Cdc48 protein complex. Once in the cytosol, translocated proteins get polyubiquitinated by E3 ubiquitin ligases marking them for proteasomal degradation. Polyubiquitinated substrates are recognized by the proteasomal degradation machinery for deubiquitylation and breakdown into peptide fragments (Ruggiano et al., 2014).

Lysosomes are double membranous cytoplasmic organelle containing a group of hydrolases that digest and breakdown macromolecules that are delivered from inside or outside the cell. Materials from outside the cell are delivered through endocytosis via clathrin coated endocytic vesicles budded from plasma membrane which will fuse with endocytic vesicles budded from the trans Golgi network (early endosomes) (Settembre et al., 2013). As a lysosomal precursor, endosomes will mature to late endosomes by lowering its pH to 5.5 preparing a suitable environment for hydrolases enzymes. Lysosomes are also involved in processing and degradation of the cell’s own macromolecules and metabolites through autophagy to maintain cellular haemostasis (Vellodi, 2005). Based on substrate uptake, autophagy is classified into three types (Singh and Cuervo, 2011). Macroautophagy occur with encapsulating denatured macromolecules or damaged organelle with membranous structures generating autophagosomes that fuses with lysosomes to initiate the digestion process. Macromolecules that follow this path are RNA, carbohydrates and polyubiquitinated-proteins, small organelle like mitochondria, and ER segments (Eskelinen and Saftig, 2009). Macroautophagy malfunctions is common in different types of LSDs like impairment fusion of autophagosome to lysosome in mucolipidosis (Fraldi et al., 2010). Lysosomes also engulf cytosolic material by pinocytosis in a process called microautophagy. It is a non-selective autophagic pathway by which lysosomes directly engulf cytoplasmic materials via membrane invagination (Li et al., 2012). Defects in such processes are not well understood yet but it has been associated with Pompe disease (PD; Takikita et al., 2009). Autophagy can be selective in internalizing its material through a receptor mediated process that only binds proteins with the KFERQ motif in chaperone mediated autophagy (CMA) (Dice et al., 1990). Mutations in lysosome-associated membrane protein 2 (LAMP-2A) and mucolipin-1 receptors causes Danon and mucolipidosis IV disorders, respectively, by affecting the CMA process and substrate uptake to lysosomes (Fidzianska et al., 2007; Venugopal et al., 2009). Cellular pathways are always linked and working together to reach haemostasis. The crosstalk between autophagy, ERS and UPR is well maintained in the cell. The UPR-PERK signaling pathway induces the activation of autophagy to get rid of aggregated proteins. Autophagy is also activated when ERAD machinery is overwhelmed or fails to efficiently degrade certain types of highly structured proteins (Senft and Ronai, 2015).

Defects in any of the cellular or biochemical mechanisms that involves hydrolase enzyme syntheses, lysosomes biogenesis, lysosome-endosome system, or lysosome-autophagy system can lead to metabolites aggregation in lysosomes, hence, the occurrence of LSDs (Platt et al., 2012). Loss-of-function mutations in the genes encoding lysosomal enzymes may disrupt total protein synthesis or misfolding and retention in the ER. The latter defects are often caused by point mutations affecting protein maturation into its active conformation, trafficking to lysosomes and handled by ERAD (Figure 1). Most of ERAD substrates might be catalytically active but structurally unstable as seen in many disorders like Gaucher and Tay-Sachs disease (Schmitz et al., 2005; Dersh et al., 2016). UPR activation in some types of LSDs especially those involving CNS degeneration, as seen in GM1-gangliosidosis mice model for example, direct cells toward programmed cell death (Tessitore et al., 2004).

Several types of LSDs are associated with Ca²⁺ signaling impairment in different organelles. In Gaucher disease for example, ER calcium channel in neuronal cells are over activated due to metabolites accumulation resulting in high flux of calcium ions out of ER (Korkotian et al., 1999). Cytosolic Ca²⁺ is also elevated in Sandhoff, Niemann–Pick A and GM1-gangliosidosis disorders as the function of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and inositol 1,4,5-trisphosphate-gated calcium channels are modulated, respectively, in these diseases (Pelled et al., 2003; Sano et al., 2009). On the other hand, alteration in mitochondrial Ca²⁺ haemostasis activates the apoptotic pathway in GM1-gangliosidosis and Mucolipidosis type IV diseases (Sano et al., 2009).

Accumulation of endolysosomes and autophagosomes have been noticed in some LSDs as a result of certain hydrolases deficiencies or substrate accumulation leading to the accumulation of more substrates and impairment of lysosomal pathways like the sphingolipid pathway in multiple LSDs (Prinetti et al., 2011). Alterations in the autophagy function contribute to the pathogenesis of many LSDs which leads to the accumulation of autophagosomes in affected cells and activation of cell death pathways. In GM1-gangliosidosis, neuronal ceroid lipofuscinosis, and Niemann–Pick type C disorders, overactivation of autophagy has been observed while in multiple sulfatase deficiency and mucopolysaccharidosis type IIIA (MPSIIIA) disorders autophagosome fails to fuse with lysosomes (Vitner et al., 2010).

Diagnostic process of LSDs remains a challenge to many clinicians due to the clinical overlap of signs and symptoms between different disorders in combination with their rarity leading to misdiagnosis or significant delay of diagnosis (Kishnani et al., 2013). Most LSDs are usually presented with an early loss of acquired cognitive and motor skills in addition to various symptoms affecting different organs such as the spleen and liver (Wilcox, 2004). Patients’ may show neurological symptoms in combination with cardiac, musculoskeletal, and/or ophthalmologic features like corneal clouding (Staretz-Chacham et al., 2009). The correct diagnosis is a result of a productive collaboration between specialized clinicians and laboratory specialists. Generally, LSDs diagnosis is based on three major stages including preliminary clinical screening, biomedical testing, and genetic molecular testing (Filocamo and Morrone, 2011).

Patients are first assigned for clinical screening of the presented signs and symptoms. There are some common diagnostic features that have been clinically associated with specific disorders (Kingma et al., 2015). Features like cerebellar ataxia in GM1/2 gangliosidosis, Neuropathic pain and kidney failure in Fabry disease, Seizures and deafness in Krabbe, Hydrops fetalis in Farber, galactosialidosis, Gaucher, GM1 gangliosidosis and others, and ocular anomalies in Farber, galactosialidosis, GM1/2 gangliosidosis, sialidosis, and Gaucher (Kingma et al., 2015). Kingma et al. (2015) has suggested a diagnostic algorithm for a group of LSDs presented with dysmorphia, musculoskeletal manifestations and/or progressive cognitive impairment. Diagnosis is generally based on urine analyses of accumulated metabolites where glycosaminoglycans (GAGs) can be an indication of mucopolysaccharidoses (MPS) or multiple sulfatase deficiency. Sialic acid in urine is a common presentation of sialic acid storage disease while, oligosaccharides can be found in patients’ with GM1/2 gangliosidosis, fucosidosis, galactosialidosis, mannosidosis, and sialidosis. Diagnosis based on urine analysis carries a risk of false negative results in cases with mild loss of enzymatic activities as seen in MPS III or MPS IV. It is very important to know that LSDs are heterogenic with wide spectrum of signs and symptoms that may vary within the same disorder and therefore diagnosis based on clinical presentation only is often inconclusive and further analyses are required.

Measurement of residual enzymatic activities of different lysosomal enzymes are crucial in the diagnosis of primary LSDs. Enzymatic assays can be performed in various types of tissues expressing the targeted enzyme like serum, leukocytes, fibroblasts, and urine. Such assays are mostly fluorometric or colorimetric using artificial tagged substrates. The complete or excessive loss of enzymatic activity is enough to confirm the underlying diagnosis but in cases with normal enzymatic activities that are presented with clinical symptoms genetic testing is needed (Filocamo and Morrone, 2011).

Recently, genetic testing using whole-exome sequencing (WES) and whole-genome sequencing (WGS) have been successfully used in clinical diagnostics of many disorders including LSDs due to their fast, accurate and reduced costs (Katsanis and Katsanis, 2013). Analysis of DNA or RNA for mutations is required to identify LSDs with non-enzymatic lysosomal protein defects and it is used to confirm the results obtained from the enzymatic activity measurements (Platt et al., 2012). In post-mortem diagnoses, genetic testing is the best diagnostic approach because the only sample that can be collected is DNA. Results from genetic testing should be interpreted carefully as some genetic changes may just be polymorphisms as seen with c.1151G > A (p.S384N) variation in Maroteaux–Lamy syndrome (Zanetti et al., 2009).

Treatments for LSDs are mainly directed toward relieving the disease symptoms through supportive medical therapies. The first implemented therapy to correct the causative defect of the underlying disorder was in the 1990s (Parenti et al., 2013). Understanding the pathophysiology underlying LSDs have profound multiple therapeutic implications to increase the lost residual enzyme activities in different LSDs. In most LSDs, 10% of the residual enzyme activity is sufficient to enhance patients’ clinical presentation as well as only cells within the affected tissues need this enzyme enhancement for therapeutic benefits, unlike many other monogenic disorders that might require recovery in all tissues and organs. Based on LSDs pathophysiology, therapies are dedicated to either compensate for the enzyme loss or reduce the accumulated substrates.

As previously mentioned, hydrolases enzymes are transported to lysosomes via M6P receptors which are also expressed on cellular plasma membranes. A small portion of the ER synthesized enzymes are directly secreted extracellularly but are recaptured and internalized via the membranous M6P receptors to be delivered to lysosomes through the secretory pathway (Coutinho et al., 2012). Based on lysosomal cell biology, LSD deficient cells can take up exogenous enzyme through the M6P recapture mechanism. Therefore, therapies such as ERT, gene therapy, and stem cell transplant restore some of the lost enzyme using this principle.

Pharmaceutical chaperone therapy is considered a promising therapeutic approach for LSDs with conformational defects. However, there are still several major challenges for this approach to be fully translated into clinics. For example, this approach is mainly suitable for mutations causing the loss of function due to misfolding or trafficking of the protein. It is not suitable for mutations affecting residues in the active site or mutations affecting protein expression (Suzuki, 2014). To date, PCs are known to have mutation specific effect as not all missense mutations are responsive for a single compound (Takai et al., 2013; Hossain et al., 2015). Another major challenge in developing PCs as therapeutics is their specificity, especially with active site competitive inhibitors. For example, due to the structural similarities between GALC and B-Gal active sites, obtaining specific PCs for these enzymes is challenging (Deane et al., 2011). Additionally, high dosages of competitive inhibitor PCs may have adverse effects on enzymatic activities and hence loss of the target enzyme function. A suggested solution is to give the patient on PC therapy breaks and avoid continuous treatment (Ringe and Petsko, 2009). To enhance chaperones specificity, non-active site inhibitors have been developed but allosteric PCs showed significant off-target effects in some cases. Adjusting PC concentrations can be a solution to overcome the off-target effects (Valenzano et al., 2011). It has been suggested that combination therapy of PCs with proteostasis regulatory compounds or ERT can be a suitable solution for this challenge (Pastores, 2010; Kirkegaard, 2013). Finally, PCs efficacy is mainly measured by increase enzymatic activity which can be misleading in some cases. Different assays must be developed to measure different parameters affecting PC therapy (Spratley and Deane, 2016). PRs on the other hand act generally on different proteostasis pathways and are not specific for lysosomal proteins. The major consequence of the non-specific modulation of these pathways is the induction of ERS and UPR which can affect cellular function or viability (Song et al., 2013).

Undoubtedly, there is an urgent need to find medical solutions and therapies for the untreatable as well as unsatisfactorily managed LSDs. LSDs are very diverse group of disorders with broad range of symptoms, distinct defected enzymes, and accumulated metabolites. The extensive understanding of proteostasis, lysosomal pathways, and pathophysiology of LSDs will aid in the development of novel approaches in LSDs treatments including precision and personalized therapies. Although therapies using PCs and protein regulators are still in their early stages, it showed promising results in some LSDs as well as the combination use of these compounds with conventional therapies showed enhanced clinical outcomes. Therefore, extensive studies are required to fully elucidate and understand the biological and cellular mechanisms of the LSDs-causing mutations as well as expand the search for small molecules to correct some of the underlying cellular defects as novel therapies for these life-threatening conditions.

FM searched the literature, wrote the draft of the manuscript and prepared the figures. BA initiated the project, edited and supervised the overall progress of the manuscript. FA-J and LA-G revised the manuscript. All authors revised and approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.