The internalization and processing of cargo via the endocytic pathway is a complex process involving numerous steps that include cargo binding and entry, receptor–ligand dissociation, membrane fusion, degradation of internalized lipids, proteins, and carbohydrates, and excretion of digested products through the lysosomal membrane (45
). The efficiency of these processes depends on the maintenance of specific ionic environments that are established by the combination actions of ion channels and pumps residing in endocytic compartments (32
). New information about these ion transporters regularly emerges from studies on the genetic determinants of lysosomal storage diseases. MLIV is one of several examples of lysosomal storage diseases caused by a dysfunction in the ionic balance of the endocytic machinery. The severity of this disease is compounded by the fact that no unified model exists for MLIV pathogenesis, thus hindering the development of pharmacological interventions.
Because the identification that mutations in the MCOLN1
gene are responsible for the MLIV disease phenotype (1
), it has become apparent that this lysosomal cation channel plays a key role in the maintenance and regulation of postendocytic events (5
). As described in the Introduction, two models exist to explain the primary defect in MLIV. The biogenesis model suggests that MLIV is caused by delayed membrane traffic to lysosomes as a result of impaired endosome–lysosome fusion or fission (21
). The metabolic model postulates that MLIV results from an ionic imbalance in lysosomes that precludes efficient processing of internalized lipids and other molecules (13
In principle, a comprehensive model for the regulation of TRP-ML1 channel activity would enable the discrimination between these models. Fusion between various membrane-bound vesicular endocytic compartments is regulated by Ca2+
concentration in the vicinity of these compartments, whereas the trafficking and activity of various proteases and lipases depends on the acidic environment maintained within both endosomal and lysosomal compartments (32
). TRP-ML1 has been reported to be permeable to both Ca2+
and to H+
; however, electrophysiological characterization of TRP-ML1 activity under physiological conditions has yet to be performed (13
In the present studies, we sought to determine whether MLIV results from aberrant membrane traffic to lysosomes. An additional issue is whether any observed defects in membrane trafficking or lipid metabolism are a primary result of MLIV pathophysiology, or are instead secondary effects that result from the chronic accumulation of undigested lipids in TRP-ML1–deficient cells. Our goal was to design an experimental system in which we could examine these membrane-trafficking steps in cells that lacked TRP-ML1 but that had not yet chronically accumulated undigested lipids. Because MLIV has been unequivocally identified as a “single-gene disorder”, acute TRP-ML1 knockdown using siRNA is a valid model for the early stages of MLIV pathogenesis. Using this approach, we were able to down-regulate TRP-ML1 expression in cells over a period of several days. During this time, lysosomes from these cells gradually accumulated lipid inclusions, but at a level significantly less than previously described in MLIV fibroblasts (13
). Formation of inclusions was efficiently rescued by transfection of an siRNA-resistant version of TRP-ML1. Lipids accumulated in lamp-1–positive organelles, suggesting that the inclusions are of lysosomal origin. Furthermore, colloidal gold internalized by fluid-phase endocytosis could access inclusions in both isolated patient fibroblasts and in siRNA-treated cells, demonstrating that these organelles remain active components of the endocytic pathway.
Several studies have reported that the trafficking of fluorescent conjugates of LacCer is hindered in MLIV fibroblasts as well as in other lysosomal storage diseases (13
). Consistent with these observations, we observed a similar defect in LacCer handling in cells treated with TRP-ML1–specific siRNA. However, we observed no difference in the delivery of this lipid to lysosomes in control and TRP-ML1 knockdown cells. Therefore, the altered trafficking of LacCer may represent an effect on postlysosomal membrane traffic and/or lipid metabolism in cells after prolonged accumulation of undigested materials. These results are consistent with a recent report by Thompson et al. (43
) suggesting that the primary defect in MLIV lipid handling may be the exit of internalized lipids from lysosomes. We found no effect of acute TRP-ML1 knockdown on the rate of LDL delivery to lysosomes or on the degradation of both the lipid and protein components of this complex. Collectively, these data argue strongly against the biogenesis model for MLIV progression, which predicts that acute loss of TRP-ML1 function will result in a global defect in the delivery of both internalized lipids and proteins to lysosomes.
The role of TRP-ML1 in lysosomal ion homeostasis is currently disputed. Increased accumulation of acridine orange in MLIV fibroblasts has previously been observed (13
), consistent with increased acidity of these organelles; however, quantitation of lysosomal pH in MLIV fibroblasts by other groups has yielded discrepant results (21
). Similar to Pryor et al. (21
), we were unable to document any difference in lysosomal pH between control and MLIV fibroblasts by fluorescence ratio imaging; however, our studies were compromised by the intense autofluorescence in MLIV lipid inclusions that has previously been reported (41
). However, we reproducibly observed that lysosomal pH in HeLa cells lacking functional TRP-ML1 was more acidic than control (a decrease of ~1.12 pH units). In principle, this finding is consistent with the metabolic model for MLIV pathogenesis, which postulates that the increased acidity of lysosomes disrupts lipid hydrolysis.
Previous groups have demonstrated defects in lipase handling in MLIV fibroblasts, including delayed deesterification of cholesterol esters and decreased lysosomal acid lipase activity (13
). Consistent with this, we found that the release of free fatty acids from [14
C]CO-labeled LDL was slowed in MLIV fibroblasts. Surprisingly, however, we did not detect a deficit in cholesterol metabolism in siRNA-treated cells (after 1, 5, or 12 d).
The apparently normal hydrolysis of LDL cholesterol upon acute loss of TRP-ML1 function demonstrates that the increased lysosomal acidity observed in these cells does not critically impair acid lipase activity. Rather, there appears to be a gradual effect on lysosomal hydrolysis that manifests as a lag period between loss of TRP-ML1 function and the full elaboration of the MLIV disease phenotype. It is possible that a minor deficit in lipid hydrolysis that is undetectable early after TRP-ML1 loss has a cumulative effect whose consequences slowly develop as the disease progresses. Indeed, other lysosomal storage disease models such as Niemann-Pick type C (NPC) (48
) teach us that a defect in a single component of lysosomal machinery may sabotage processing of unrelated classes of lipids. NPC disease is caused by the mutation in either the late endosomal membrane protein NPC1 or the soluble lysosomal protein NPC2, and results in abnormal cholesterol transport along the late endocytic pathway. Similar to MLIV, NPC is caused by defective cholesterol trafficking rather than by a specific enzymatic abnormality. Although the primary defect is in cholesterol transport, the accumulation of other lipids, including sphingolipids, has also been demonstrated in NPC (48
In summary, our results suggest that TRP-ML1 does not directly regulate membrane traffic; thus, enzyme replacement therapies remain a potentially viable treatment option for MLIV. Future studies are needed to determine whether TRP-ML1 plays an essential role in maintaining lysosomal ion homeostasis directly so that replacement enzymes may be designed to operate in the unique environment of the TRP-ML1–deficient lysosome.