CLN6 MUTANTS ASSOCIATED WITH NEURONAL CEROID LIPOFUSCINOSIS ARE DEGRADED IN A PROTEASOME DEPENDENT MANNER

Abstract

Neuronal ceroid lipofuscinoses (NCL), a group of inherited neurodegenerative lysosomal storage diseases predominantly affecting children, are due to autosomal recessive mutations within one of the nine cln genes. The wild type cln gene products are comprised of membrane and soluble proteins that localize to the lysosome or endoplasmic reticulum. However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6^G123D and Cln6^M241T) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most likely generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knock down of SEL1L, a member of an E3 ubiquitin ligase complex involved in ER protein extraction rescued significant amounts of Cln6^G123D and Cln6^M241T polypeptides. The results implicate ER quality control in the instability of the Cln variants that likely contributes to the development of NCL.

INTRODUCTION

Neuronal ceroid lipofuscinoses (NCL), also referred to as Batten disease, are the most common pediatric neurodegenerative diseases having an incidence rate of 1 in 12,500 in the US population [1]. Their significant clinical feature is progressive encephalopathy confined to macular cerebral degeneration [2]. The symptoms of NCL encephalopathy include epileptic seizures, dementia, progressive psychomotor decline, visual retrocession, and blindness and ultimately leads to premature death [3]. The histological hallmark of NCL is the accumulation of proteolipid pigment deposits in lysosomes of many cell types [4, 5]. The patients suffering from Batten disease show intracellular inclusions predominately in neurons caused by lysosomal accumulation of ceroid and lipofuscin with mitochondrial ATP synthase subunit C [6].

Most NCL are inherited as autosomal recessive mutations within 6 identified and 3 unidentified genes denominated cln1-9. Defective Cln proteins result in lysosomal dysfunction and accumulation of lysosomal storage material in the brain of affected children, leading to development of several pathogenic processes and neurodegeneration. Current genetic classification of NCLs vary in clinical phenotypes from more benign to extremely severe depending on the specific genetic defect of the encoded protein. They are divided into four main groups, infantile NCL, late infantile NCL, juvenile JNCL, and adult NCL and four other less frequent entities. The Cln polypeptides are both soluble and membrane proteins located in either endosomal-lysosomal compartments (Cln1:palmitoyl-protein thioesterase, Cln2:tripeptidyl peptidase I, Cln3, Cln5) or the ER (Cln6, Cln8) [7]. Cln4, Cln7, and Cln9 proteins remain to be identified. Interestingly, the processing and fate of aberrant Cln proteins is not clear. In fact, hereditary mutations within cellular genes can result in clinically significant pathologies such as cystic fibrosis, hereditary emphysema [8], nephrogenic diabetes insipidus [9], and oculocutaneous albinism [10]. Since ER quality control has been implicated in these diseases, it is likely that it plays a role in processing of Cln variants and development of NCL.

Mutations within the cln6 gene cause both the classical late-infantile and juvenile forms of NCL [11]. Our study explores the role of ER quality control in the early processing events of ER localized Cln6 mutant proteins [12-14]. Experiments revealed that Cln6 mutant proteins were degraded in a proteasome dependent manner. In addition, knock down of SEL1L, a protein involved in disposal of aberrant ER proteins, rescued the degradation of the Cln6 mutants. The data supports a paradigm that ER quality control may contribute to the onset of NCL type 6 and offers insight towards potential therapy against NCL.

EXPERIMENTAL

Cell lines and antibodies

Human U373-MG astrocytoma cells expressing Cln6 polypeptides (U373_Cln6wt, U373_Cln6G123D and U373_Cln6M241T cells) were generated and maintained as described [15]. Anti-PDI [16], anti-SEL1L [17], and anti-calnexin antibody (AF8 [18], gift from M. Brenner (Harvard Medical School) were utilized as described. Anti-GAPDH, anti-p97, anti-Derlin-1, and anti-BiP antibodies were purchased from Chemicon International Inc., Sigma, and Stressgen, respectively.

cDNA constructs

The Cln6 chimeras were cloned from the Cln6 template (ATTC Image ID 3878776) with an amino-terminus hemagglutinin (HA) epitope tag (AYPYDVPDYA) into the retroviral vector pLpCX (Clontech). Cln6^G123D and Cln6^M241T site-directed mutant cDNAs were generated from PCR fragments [15].

Immunoprecipitation

Immunoprecipitations were performed as described [15]. In brief, cells (1× 10⁶) were lysed in 0.5% Nonident P (NP)-40 lysis mix followed by incubation with the respective antibody and protein A-agarose beads. The immunoprecipitates were resolved using SDS-PAGE (12.5%) and subjected to immunoblot analysis using the respective immunoglobulin.

Pulse-chase analysis

Pulse-chase experiment was performed as described [15]. Cells were labeled with ³⁵S-methionine and chased in cold methionine (25 mM). Cln6 proteins were recovered from NP-40 cell lysates using anti-HA antibodies and resolved using SDS-PAGE (12.5%). The polyacrylamide gel was dried, exposed to autoradiography film and polypeptides were quantified by densitometry analysis using an Alpha Imager 3400.

Immunofluorescence microscopy

Cells were fixed with methanol/acetone solution (v/v 1:1) and incubated with blocking solution (1% BSA, 0.5% cold water fish gelatin (Sigma), PBS pH 7.2) followed by the respective antibody. The cells were then incubated with the respective anti-mouse^FITC and anti-rabbit^{Texas Red} (Molecular Probes) immunoglobulins. Fluorescence microscopy was carried out using an Olympus IX70/IX-FLA inverted fluorescence microscope and a Sony DKC-5000 digital camera. Images were created with Adobe Photoshop software.

RESULTS

Cln6 mutant molecules are stabilized by inclusion of proteasome inhibition

Cln6 is a 311 amino acid non-glycosylated ER membrane protein predicted to contain either five or six transmembrane domains [12, 14]. To investigate the processing of Cln6 mutants implicated in the development of NCL, a hemagglutinin (HA) epitope tag was introduced at the amino terminus of Cln6^wt and Cln6 variants (Cln6^G123D and Cln6^M241T) with missense mutations in the predicted transmembrane domains [19]. These mutants were chosen for study because they were identified from NCL patients [12-14]. These polar residues within the membrane bilayer most likely cause the mutant to exist in a misfolded conformation and potential a target for the ER quality control apparatus. Hemagglutinin-epitope tagged versions of Cln6^wt, Cln6^G123D, and Cln6^M241T were transduced into U373 cells (U373_Cln6wt, U373_Cln6G123D, and U373_Cln6M241T cells) and the stability of the Cln6 proteins was examined by pulse-chase analysis (Figure 1A). U373_Cln6wt, U373_Cln6G123D, and U373_Cln6M241T cells were metabolically labeled for 15 min and chased for up to 45 min. Equivalent levels of wild type Cln6 polypeptides were recovered throughout the chase period (Figure 1A, lanes 1-4), a result consistent with protein stability. In contrast, levels of Cln6^G123D polypeptides rapidly decreased during the chase period (Figure 1A, lanes 5-8) with a half-life of ∼20 min (Figure 1B). Interestingly, the migration difference of Cln6^G123D versus wild type Cln6 or Cln6^M241T is probably due to a conformational change in the overall structure that is specific to the mutation. A similar result was observed in mutants of the multi-spanning membrane protein Sec61p and the soluble protein p97 [20, 21]. Even more striking was the instability of Cln6^M241T polypeptides (Figure 2A, lanes 9-12). Only a minute amount of Cln6^M241T polypeptides was recovered from the 0 min chase period and even less from the 15 min chase point suggesting a half-life of less than 15 min (Figure 2A, lane 10). These data demonstrate that Cln6 mutants are degraded with fast kinetics.

Figure 1. Cln6^G123D and Cln6^M241T are degraded with rapid kinetics.

(A) U373_Cln6wt, U373_Cln6G123D, and U373_Cln6M241T cells were pulsed with ³⁵S-methionine for 15 min and chased for up to 45 min. Cln6 polypeptides were immunoprecipitated using anti-HA antibodies and resolved on an SDS-polyacrylamide gel. The gel was exposed to autoradiography film for 3 days. The Cln6 proteins and molecular weight markers are indicated. (B) Levels of Cln6 proteins were quantified by densitometry and plotted as a percentage to the 0 min chase point (100%).

Figure 2. Cln6^G123D and Cln6^M241T proteins are stabilized in the presence of proteasome and dislocation inhibitors.

(A) Cln6 polypeptides were recovered from U373_Cln6wt (lanes 1-2, 5-6, and 10), U373_Cln6G123D (lanes 3-4), and U373_Cln6M241T (lanes 7-8) cells treated with or without proteasome inhibitor ZL₃VS. The anti-HA precipitates and total cell lysates from U373_Cln6wt and U373 cells were subjected to an anti-HA immunoblot. (B) Total cell lysates from U373_Cln6wt (lanes 1-2, 7-8, and 13-14), U373_Cln6G123D (lanes 3-4, 9-10, and 15-16), and U373_Cln6M241T (lanes 5-6, 11-12, and 17-18) cells treated with or without eeyarestatin (lanes 2, 4, and 6), lactacystin (lanes 8, 10, and 12), and ZL₃VS (lanes 14, 16, and 18) were subjected to anti-HA (lanes 1-19) and anti-GAPDH (lanes 20-38) immunoblots. U373 cell lysates were used as a negative control. The Cln6 proteins, immunoglobulin light chain (IgG LC), GAPDH, and molecular weight markers are indicated.

To examine whether the Cln6 mutants are degraded by the proteasome, Cln6 protein levels were examined from U373_Cln6wt, U373_Cln6G123D, and U373_Cln6M241T cells treated with or without the proteasome inhibitor carboxybenzyl-leucyl-leucyl-leucyl vinyl sulfone (ZL₃VS) (10μM, 5 hr) [22]. Cln6 proteins recovered from the respective cells were analyzed using an immunoprecipitation/immunoblot experiment (Figure 2A). U373 cells transduced with empty vector were used as a negative control (Figure 2A, lanes 9 and 11). Equivalent levels of Cln6^wt were recovered independently of inhibitor treatment (Figure 2A, lanes 1-2 and 5-6), whereas a minimal number of polypeptides was recovered from U373_Cln6G123D and U373_Cln6M241T cells (Figure 2A, lanes 3 and 7). However, significant amounts of Cln6^G123D and Cln6^M241T polypeptides were recovered from ZL₃VS-treated cells (Figure 2A, lanes 4 and 8). Because Cln6^G123D is degraded with slower kinetics than Cln6^M241T, we observed increased levels of Cln6^G123D than Cln6^M241T from proteasome treated cells (Figure 2A, lane 4 vs. 8). Consistent with this data, higher molecular weight, most likely ubiquitinated, Cln6^G123D and Cln6^M241T species (*) were recovered from proteasome inhibitor-treated cells (Figure S1). Collectively, these data verify that Cln6^G123D and Cln6^M241T are extracted from the ER to the cytosol and degraded by the proteasome.

To further confirm that the instability of Cln6^G123D and Cln6^M241T polypeptides was due to their transport out of the ER, we examined the effect of eeyarestatin, an inhibitor of the dislocation reaction [23]. U373, U373_Cln6wt, U373_Cln6G123D, and U373_Cln6M241T cells were treated with or without eeyarestatin (5μg/mL) or the proteasome inhibitors lactacystin [24] (10μM) or ZL₃VS (5 μM) for 12 hr. Total cell lysates were subjected to immunoblot analysis using anti-HA antibodies (Figure 2B). An anti-GAPDH immunoblot verified equal protein loading (Figure 2B, lanes 20-38). As expected, treatment with the proteasome inhibitors lactacystin and ZL₃VS caused a significant increase in both Cln6^G123D and Cln6^M241T polypeptides (Figure 2B, lanes 10, 12, 16, and 18, respectively). Moreover, eeyarestatin treatment caused increased levels of Cln6^G123D and Cln6^M241T polypeptides (Figure 2B, lanes 4 and 6), albeit to a lower level than the proteasome inhibitors. This is probably due to the inability of eeyarestatin to completely attenuate dislocation of ER substrates [23]. Eeyarestatin treatment caused a greater accumulation of Cln6^G123D than Cln6^M241T probably due to differences in their degradation kinetics (Figure 1B). In summary, Cln6^G123D and Cln6^M241T polypeptides were stabilized upon inclusion of proteasome or dislocation inhibitors.

Stabilized Cln6 mutant proteins are localized to the ER

The localization of the stabilized Cln mutant Cln6^G123D was examined using fluorescence microscopy (Figure 3). U373_Cln6wt and U373_Cln6G123D cells treated with or without ZL₃VS (2.5μM, 12 hr) were probed for Cln6 or PDI polypeptides using anti-HA (Figure 3, panels 1-2 and 7-8) and anti-PDI (Figure 3, panels 3-4 and 9-10) antibodies, respectively. The merged images represent the co-localized polypeptides (Figure 3, panels 5-6 and 11-12). The intensity of the Cln6^wt fluorescence signal was independent of proteasome-inhibitor treatment (Figure 3, panels 1-2) and co-localized with the ER resident protein PDI (Figure 3, panels 5-6). In contrast, a significant increase in the fluorescence signal was observed in U373_Cln6G123D cells treated with proteasome inhibitor (Figure 3, compare panels 7 and 8). The ER localization of Cln6^G123D polypeptides upon proteasome inhibition was confirmed by their co-localization with PDI (Figure 3, panel 12). In addition, a subcellular fractionation experiment [25] revealed that the majority of Cln6^G123D and Cln6^M241T mutant polypeptides was observed from the membrane fraction upon proteasome inhibitor treatment (Figure S2). These results demonstrate that stabilized Cln6 mutants are localized to the ER.

Figure 3. Stabilized Cln6 mutants localize to the ER.

U373_Cln6wt (panels 1-6) and U373_Cln6G123D (panels 7-12) cells treated with or without proteasome inhibitor ZL₃VS were probed with anti-HA (panels 1-2 and 7-8) and anti-PDI (panels 3-4 and 9-10) antibodies and subjected to indirect immunofluorescence microscopy. The yellow regions in the merged images (panels 5-6 and 11-12) represent the co-localized material.

Cln6^G123D and Cln6^M241T polypeptides interact with the dislocation complex

The dislocation of ER proteasomal substrates is mediated through the interaction with both ER and cytosolic proteins. Derlin-1, an ER membrane protein essential for dislocation of certain ER degradation substrates, together with the cytosolic complex of AAA ATPase p97/Ufd1/Npl4 facilitates the extraction of ER proteins out of the ER [26]. To further define the processing of the Cln6^G123D and Cln6^M241T mutants, we examined whether Cln6^G123D and Cln6^M241T complex with the specific components of the dislocation apparatus, Derlin-1 and p97. Derlin-1 was recovered from U373_Cln6wt, U373_Cln6G123D,and U373_Cln6M241T cells treated with or without ZL₃VS (10 μM, 5 hr) using anti-Derlin-1 antibodies. The immunoprecipitates and total cell lysates were subjected to an anti-HA (Figure 4A, lanes 1-14) and anti-Derlin-1 (Figure 4A, lanes 15-28) immunoblot. As expected, Derlin-1 polypeptides were recovered from all samples (Figure 4A, lanes 15-28). Consistent with previous data (Figure 2), Cln6^G123D and Cln6^M241T were stabilized in the presence of proteasome inhibitor (Figure 4A, lanes 11 and 13). Both Cln6^G123D and Cln6^M241T polypeptides co-precipitated with Derlin-1 molecules from proteasome inhibitor-treated U373_Cln6G123D and U373_Cln6M241T cells (Figure 4A, lanes 4 and 6). The lack of a polypeptide signal from U373 and U373_Cln6wt cells (Figure 4A, lanes 1-2 and 7) confirmed the specificity of the anti-Derlin-1 immunoprecipitation. These results suggest that Derlin-1 is probably involved in the dislocation of Cln6^G123D and Cln6^M241T mutants.

Figure 4. Cln6 mutants complex with the dislocation apparatus.

(A) Derlin-1 was recovered from U373, U373_Cln6wt, U373_Cln6G123D, and U373_Cln6M241T cells treated with or without proteasome inhibitor ZL₃VS using anti-Derlin-1 antibodies. The precipitates (lanes 1-7 and 15-21) and total cell lysates (lanes 8-14 and 22-28) were subjected to immunoblot analysis using anti-HA (lanes 1-14) and anti-Derlin-1 (lanes 15-28) antibodies. Cln6 proteins were recovered from U373_Cln6wt (B and C), U373_Cln6G123D (B), and U373_Cln6M241T (C) cells treated with or without proteasome inhibitor ZL₃VS. The precipitates and U373_Cln6wt total cell lysates (B and C) were subjected anti-p97 (B and C, lanes 1-5) and anti-HA (B and C, lanes 6-10) immunoblots. Derlin-1, p97, Cln6 proteins, and molecular weight markers are indicated.

We next examined a possible interaction between Cln6^G123D and Cln6^M241T and p97 (Figure 4B and C). Cln6 polypeptides recovered from U373_Cln6wt, U373_Cln6G123D, and U373_Cln6M241T cells treated with or without ZL₃VS (5 μM, 12 hr) were subjected to immunoblot analysis. As expected, significant amounts of Cln6^G123D and Cln6^M241T proteins were recovered from the proteasome inhibitor treated cells (Figure 4B and C, lane 9). p97 exclusively co-precipitated with Cln6^G123D and Cln6^M241T from ZL₃VS-treated cells (Figure 4B and C, lane 4) and not with Cln6^wt (Figure 4A and B, lanes 1-2). Collectively, these results suggest that Derlin-1 and p97 probably mediate the extraction of Cln6^G123D and Cln6^M241T polypeptides from the ER and into the cytosol.

Cln6^G123D and Cln6^M241T are dislocated in a SEL1L dependent manner

Ubiquitination of ER degradation substrates through a specific E3 ligase is a prerequisite for the dislocation of most degradation substrates [27]. In fact, ER localized membrane proteins that complex with E3 ligases play an important role in the dislocation process. Sel-1 suppressor of lin-12-like (SEL1L), an ER membrane protein that complexes with the E3 ligase HMG Co-A reductase degradation protein 1 (Hrd1), Derlin-1, Derlin-2, and p97 [28, 29] has been implicated in the dislocation of both membrane and soluble degradation substrates [17, 30]. Is SEL1L involved in the degradation of Cln6^G123D and Cln6^M241T? To address this question, we examined the steady state levels of Cln6 polypeptides in cells that stably express shRNA against SEL1L (shRNA-SEL1L) or GFP (shRNA-GFP) [17]. The shRNA-GFP cells were utilized as a negative control. U373_Cln6wt, U373_Cln6G123D, and U373_Cln6M241T cells expressing shRNA-SEL1L or shRNA-GFP were treated with proteasome inhibitor ZL₃VS (10 μM, 5 hr). The total cell lysates were subjected to immunoblot analysis to determine the levels of Cln6 polypeptides (Figure 5). Cln6^wt levels were not altered upon inclusion of proteasome inhibitor or expression of shRNAs (Figure 5, lanes 1-3 and 10-12). As expected, in shRNA-GFP expressing cells, the levels of Cln6^G123D and Cln6^M241T increased upon the treatment with proteasome inhibitor (Figure 5, lanes 4-6 and 7-9, respectively). Strikingly, significant levels of both Cln6^G123D and Cln6^M241T polypeptides were observed from SEL1L knock down cells (Figure 5, lanes 13-18). Since SEL1L was knocked down only ∼50% (Figure S3), a greater attenuation of Cln6^G123D and Cln6^M241T degradation would be probably observed upon a complete knock down of SEL1L. The GAPDH immunoblot confirmed equal loading of cell lysates (Figure 5, lanes 20-38). These results strongly support the hypothesis that Cln6^G123D and Cln6^M241T degradation is dependent on SEL1L and is regulated by ER quality control.

Figure 5. Knockout of SEL1L in cells attenuates Cln6^G123D and Cln6^M241T degradation.

Total cell lysates from U373_Cln6wt, U373_Cln6G123D, and U373_Cln6M241T cells that express shRNA against either GFP (lanes 1-9 and 20-28) or SeL1L (lanes 10-19 and 29-38) were treated with proteasome inhibitor ZL₃VS. The samples were subjected to immunoblot analysis using anti-HA (lanes 1-19) and anti-GAPDH (lanes 20-38) antibodies. The Cln6 proteins, GAPDH, and molecular weight markers are indicated.

DISCUSSION

NCL are neurodegenerative childhood diseases caused by mutations within 9 different cln genes [31]. Cln mutants are either truncated versions of the protein or contain point mutations yielding enzymatically inactive polypeptides or molecules that never reach their final destination. The early processing events within the ER can dictate the fate of these mutant proteins. In fact, specific genetic diseases including cystic fibrosis, emphysema, Fabri disease, Gaucher disease, GM1 Gangliosidosis, and Tay-Sachs disease are caused by mutations within the proteins that are subjected to ER quality control [32, 33]. The present study shows for the first time that the Cln6 mutants Cln6^G123D and Cln6^M241T implicated in the onset of NCL type 6 are unstable and degraded in a proteasome dependent manner. Hence, the development of NCL may be attributed to the instability of the mutant polypeptide implying that ER quality control contributes to the onset of disease.

ER quality control has evolved to prevent the egress of defective membrane or secretory polypeptides from the ER. In most cases, the misfolded proteins are recognized by ER chaperones such as BiP, calnexin, and PDI [34, 35] and extracted through the ER membrane via a proteinaceous channel to be degraded by the proteasome. The recruitment of the degradation substrate to the dislocon occurs through an interaction with membrane-bound ubiquitin-conjugating enzymes (i.e. Ubc7, Ubc1, Ubc6), E3 ubiquitin ligases (i.e. gp78, Hrd1/Der3, Doa10, HsHrd1, trc8, and TEB4) [27], and factors such as the Derlin family of proteins and SEL1L. Once at the site of dislocation, p97 complexed with Npl4 and Ufd1 extracts the ubiquitin-conjugated dislocated substrate through the membrane [29, 36]. The N-linked glycan attached to the polypeptide would then be efficiently removed followed by deubiquitination and degradation by the proteasome. The mutant Cln6 proteins are degraded as classical misfolded proteins as observed by their association with ER chaperones calnexin and BiP (data not shown) as well as with Derlin-1 and p97 (Figure 4). Since the knock down of SEL1L caused an increase in levels of Cln6 mutants, it is possible that SEL1L contributes to the instability of Cln6^G123D and Cln6^M241T proteins. Collectively, the data is consistent with the paradigm that Cln6^G123D and Cln6^M241T mutant proteins are recognized and disposed of by the ER dislocation/degradation apparatus.

The Cln6^G123D and Cln6^M241T mutants are degraded with fast kinetics (Figure 1). Given the fact that the Cln6 protein spans the membrane seven times, we would expect that the degradation kinetics would be similar to the degradation kinetics of CFTR mutants [37]. Why are these aberrant proteins degraded in such a fast manner? Since both Cln6 mutations lie within the transmembrane regions, the amino acid changes may disrupt the stability of the transmembrane domains and induce a drastically misfolded protein that is quickly discard for degradation by the proteasome. The severity of the misfolded state of the protein can directly dictate the half-life of the polypeptide. Therefore, Cln6^G123D would be in a more native state than Cln6^M241T. Nevertheless, the dramatic instability of the Cln6 mutants can prove to be a useful model system to study how multi-membrane spanning proteins are dislocated across the ER membrane.

The degradation of aberrant Cln6 polypeptides implies that those proteins are thwarted in their cellular function that eventually leads to the accumulation of insoluble proteolipid material in the lysosomes. It remains to be clarified whether increasing the stability of these mutants would allow for the Cln mutant to regain at least partial wild type activity. Therefore, proteasome inhibitors may contribute to the considered as potential therapeutics for NCL type 6 if the stabilized Cln6 mutant would retain at least partial activity. In fact, understanding the process of ER quality control in the development of specific genetic diseases has already given promising results in the treatment of Fabri disease caused by mutations in alpha-galactosidase polypeptides. Namely, 1-deoxygalactonojirimycin, a competitive inhibitor of alpha-galactosidase could stabilize alpha-galactosidase mutant and correct its trafficking defect [38]. In addition, chemical chaperones could rescue some lysosomal storage diseases caused by inherited mutations such as in Gaucher disease, GM1 Gangliosidosis and Tay-Sachs disease [39-41]. The current study serves as a preliminary guide for identifying potential new treatments against NCL that could be examined in the available animal models. A number of spontaneous animal forms of NCL have been described including sheep, dogs, and mice [42-44]. The mouse models of NCLs, Ppt1^−/− [45], Ppt1^Δex4 knockout [46], Tpp1 neo^insArg446His knockout [47], Cln5^−/− knockout [48], Cln6^nclf spontaneous mutant [49], Cln8^mnd spontaneous mutant [50], Cln3^−/− knockout [51], Cln3^Δex7/8 knock-in [52], Cln3 knockout [53] demonstrate phenotypes that closely resemble the major features of human NCLs. They all show clinically relevant behavioral and pathological changes that exhibit a progressive neurodegenerative disorder. The value of animal models for neurodegenerative disorders lies largely in the utility of these models for evaluating potential therapeutic interventions in the future [54]