WO2013070981A2 - Treating er stress related disorders by stabilizing intracellular calcium homeostasis - Google Patents

Abstract

Disclosed herein are methods of treating and preventing neurodegenerative disorders associated with calcium imbalances in neural cells. Provided are compositions that serve to stabilize calcium homeostasis. Particularly exemplified is the administration of salubrinal for a subject in need of treatment.

Description

TREATING ER STRESS RELATED DISORDERS BY STABILIZING INTRACELLULAR

CALCIUM HOMEOSTASIS

CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional patent application no. 61 /704,074 to which priority is claimed under 35 USC 1 19. This application is incorporated herein in its entirety by this reference.

BACKGROUND

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that results from the degeneration of dopaminergic (DA) neurons in the substantia nigra (1 ). A common pathological feature of PD is the aggregation of a-synuclein (aSyn) into cytoplasmic inclusions called Lewy bodies in the degenerating dopaminergic neurons (1 ). Cell culture studies have shown that overexpression, impaired turnover, and mutations lead to aSyn aggregation (2). Two missense mutations (Ala53Thr and

Ala30Pro) in aSyn that cause early-onset, autosomal dominant forms of PD enhance the aggregation and toxicity of the protein (2). Duplication or triplication of the aSyn gene was also found to cause early onset PD suggesting that elevated levels of wild- type aSyn can also lead to neurotoxicity (3). It is not yet clear how aSyn aggregation induces the degenerative cascades leading to PD. Recent studies have demonstrated that mutant aSyn may exert its pathological effects in parts by inactivating the Grp78/Bip chaperone function (4) or impeding endoplasmic reticulum (ER) to Golgi vesicular transport (5) leading to abnormal accumulation of proteins within the ER and induction of ER stress. Cells respond to ER stress by activating the unfolded protein response (UPR) aimed at inducing translational repression and expression of ER-resident chaperones to enhance protein folding, processing and degradation of misfolded proteins, thus relieving cells from ER stress (6). Prolonged or unmitigated ER stress associated with insufficient degradation of misfolded proteins or deranged calcium (Ca²⁺ ) homeostasis would subsequently activate ER stress-associated apoptotic pathways (7).

Hallmarks of ER stress are detected in several experimental models of PD (8, 9) and in nigral dopaminergic neurons of PD subjects (10). Expression of PD-linked mutant aSyn elevates CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) (1 1 ), an ER stress-induced apoptotic mediator (12). CHOP is also elevated in

neurotoxin models of PD (8, 9) and is a critical mediator of apoptotic death in substantia nigra dopamine neurons (1 1 ,13). Furthermore, ER stress is associated with the aggregation of aSyn in dopaminergic neurons (10). Notwithstanding these studies, the underlying mechanisms of ER stress-mediated degenerative cascades and the specific roles of the various UPR proteins in PD pathogenesis remain unknown.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Expression of mutant aSyn induces a heightened ER stress response (A) A representative immunoblot showing the time course of wild-type (WT) human aSyn protein level after the addition of Tet. The antibody used was specific for human aSyn. (B) Representative gel images (top) and immunoblots (bottom) of WT and mutant (A30P and A53T) aSyn, Herp, Grp94/78, and CHOP mRNA and protein levels, respectively, in PC12 cells 72 h after the addition of Tet (induced) or vehicle (non- induced). PCR products amplified were separated on ethidium bromide stained agarose gels. Blots were reprobed with ERK1 to confirm equality of total protein loading. (C) Representative immunoblots (top) and results of densitometric analysis (bottom) of Herp, Grp94/78, and CHOP protein levels in PC12 cells at the indicated time points following transient transfection with either WT or mutant (A53T) aSyn. Control cells were transfected with the empty vector (VT). The antibody used was specific for human aSyn. Values are the mean ± SEM of three independent experiments. #p<0.05;

*p<0.01 , compared to PC12-VT and PC12-WTa-Syn and between the indicated groups.

FIG. 2: Herp protects from mutant aSyn-induced cell death

(A) Trypan blue exclusion was used to determine the viability of the indicated PC12 clones at 96 h under induced or non-induced conditions. Data represent the mean ± SEM of three separate experiments. #p<0.05,^*p<0.01 , compared to PC12-WTaSyn and PC12-VT under non-induced and induced conditions; ^*p<0.01 , between the indicated clones expressing mutant aSyn. (B) Representative immunoblots of protein levels of aSyn and CHOP and caspase-12 (Casp12) processing in the indicated PC12 cells 72 h after the addition of Tet (induced) or vehicle (non-induced). Appearance of the active proteolytic fragment of Casp12 is indicated by an asterisk. ERK is used as an internal control of protein loading. Histogram shows densitometric analysis of CHOP protein. #P<0.05, versus the PC12-WTaSyn and PC12-VT under non-induced and induced conditions; (C) Histograms show the viability of the PC12-A53T after transfection with siRNA-Con and siRNA-Herp (100 nM). One day after transfection, Tet was added to the cultures and cell viability was determined 48 h after by trypan blue exclusion. Values represent the mean ± SEM of three separate experiments. #p<0.05, ^*p<0.01 compared to respective non-induced and between the indicated induced PC12 cells. (D)

Representative immunoblots of aSyn, Herp and AUBL-Herp protein levels in the indicated PC12 cells under non-induced (-) or induced (+) conditions for 48 h. PC12 cells were transduced with viral particles expressing empty vector or vector containing Herp or AUBL-Herp construct 48 h prior to induction. ERK is used as an internal control of protein loading. (E) Histograms show the viability of the indicated PC12 cells after ectopic expression of Herp and AUBL-Herp. Trypan blue exclusion was used

todetermine cell viability 48 and 96 h after induction. Values represent the mean ± SEM of three separate experiments. #p<0.05, ^*p<0.01 , compared to groups transduced with the empty vector or Herp and between the indicated transduced groups.

FIG. 3: ER stress-induced by tunicamycin and mutant a-Syn perturbs ER Ca²⁺ homeostasis through the aberrant accumulation of ER-resident Ca²⁺ release channels

(A) Representative recordings of the bradykinin (BK; 10 M)-induced elevation of intracellular Ca2+ ([Ca2+]i) in PC12 cells 48 h after expression of wild-type (WT) and mutant (A53T) aSyn. PC12 cells transfected with empty vector (VT) were included as controls. Cells were loaded with fura-2 and [Ca²⁺ i was recorded in Ca2+ free medium as described under "Materials and Methods". Arrow indicates time of BK addition.

Histograms show Ca2+ peak values (change from baseline) and AUC (area under the curve). Values are the mean ± SEM of determinations made in 4 to 6 separate cultures (15-20 cells assessed/culture). #p<0.05, compared to VT. (B) Histograms show the percent of viable cells after treatment of PC12-Tuni (top) and PC12-A53TaSyn (bottom) with the indicated doses of BAPTA-AM. PC12 cells were pretreated with BAPTA-AM 2 h prior to either exposure to Tuni (20 pg/mL) or expression of VT, WT and A53T. Cell viability was determined 24 h after by Trypan blue exclusion. Values represent the mean ± SEM of three separate experiments. ^*p<0.01 , #p<0.05, compared to vehicle or VT at each time point and between the indicated groups. (C) Representative

immunoblots(7eft) and results of densitometric analyses (right) of IP3R1 , pan-RYR and PS1 protein levels in PC12-Tuni at the indicated time points. Values represent the mean ± SEM of three independent experiments. ^*p<0.01 , #p<0.05 compared to the untreated group. (D) Representative immunoblots (left) and results of densitometric analyses (right) of a-Syn, IP3R1 and PS1 protein levels in PC12 cells at the indicated time points after expression of VT, WT or A53T. Values represent the mean ± SEM of three separate experiments. #p<0.05, ^*p< 0.01 , compared to VT and WTaSyn. (E) A representative immunoblot (top) and results of densitometric analysis (bottom) of pan- RYR protein in PC12 cells at the indicated time points after expression of VT, WT orA53T. The Tuni-treated samples were included as positive controls for ER stress- induced increase of pan-RyR protein level. ^*p<0.01 , compared to VT and WT. Equal protein loading in the immunoblots shown in C-E was confirmed after reprobing the membranes for actin.

FIG. 4: Pharmacological inhibition or gene knockdown of ER-resident Ca²⁺ release channels ameliorates ER stress-induced cell death

(A) Histograms show the percent of viable PC12 cells after treatment with tunicamycin (Tuni; 20 μg/ml) (+) or vehicle (-) for 24 h in the absence or presence of the indicated doses of xestospongin C (an IP3R blocker) or dantrolene (a RyR blocker). Both drugs were added 2 h prior to Tuni. Values are the mean ± SEM of three independent experiments. #p<0.05; ^*p< 0.01 , compared to vehicle control and between the indicated groups. (B) Histograms show the percent of viable PC12 cells expressing empty vector (VT), wild-type (WT) and mutant (A53T) a-Syn. Xestospongin C (5 μΜ) or dantrolene (50 μΜ) were added 24 h after induction. Values are the mean ± SEM of three independent experiments. #p<0.05; ^*p< 0.01 , compared to VT and WTa-Syn and between the indicated groups. (C) Histograms show the fold change of the indicated ER Ca²⁺ release channel, IP3R1 (top) or RYR1 /RYR3 (bottom), in PC12 cells 24 h after transfection with the respective siRNAs (left panels) and the percent of viable

transfected PC12 cells at the indicated time points after exposure to Tuni or vehicle (Con) (right panels). The siRNAs were added either 8 or 24 h (denoted by asterisk) prior to Tuni exposure. Values represent the mean ± SEM of three independent experiments. #p<0.05;^*p< 0.01 , compared to Con and between the indicated groups. (D) Histograms show the viability of the indicated PC12 cells in the presence of siRNA-IP3R1 (top) or siRNA-RYR1 and siRNA-RYR3 combined (bottom). Values represent the mean ± SEM of three independent experiments. #p<0.05; ^*p<0.01 , compared to siRNA-Con

FIG. 5: Salubrinal ameloriates the induction of ER stress markers and levels of ER-resident Ca²⁺ release channels

(A) Representative immunoblots (top) and results of densitometric analysis (bottom) of the indicated protein levels in PC12 cells that were treated with Salubrinal (Sal; 75 μΜ) for 2 h prior to exposure to tunicamycin (Tuni; 20 μg/ml). Cells were collected at the indicated time points for immunoblotting. Values represent the mean ± SEM of three independent experiments. ^*p<0.01 , compared between the indicated groups. (B) Representative immunoblots (top) and results of densitometric analysis (bottom) of the indicated protein levels in PC12 cells expressing empty vector (VT), wild-type (WT) or mutant (A53T) aSyn after treatment with Sal (+) or vehicle alone (-). Cells werecollected 24 h after Sal treatment. Values represent the mean ± SEM of three independent experiments. ^*p<0.01 , compared between the indicated groups. (C) qRT-PCR analysis of the relative expression of the indicated ER Ca²⁺ release channels in PC12 cells that were treated with Sal for 2 h prior to exposure to Tuni for the indicated time points.

FIG. 6: Herp stabilizes Ca²⁺ homeostasis by preventing ER stress-induced accumulation of ER-resident Ca²⁺ release channels

(A) Representative recordings of the bradykinin (BK)-evoked increase of intracellular Ca²⁺ ([ Ca²⁺ ]i) in PC12 cells expressing mutant (A53T) aSyn 24 h after transfection with siRNA-Con or siRNA-Herp (100 nM). Arrow indicates the time of BK addition. Cells were loaded with fura-2 and [Ca²⁺ ]i was recorded in Ca²⁺ free medium as described under "Materials and Methods". Histograms show Ca²⁺ peak values (change from baseline) and AUC (area under the curve). Values are the mean ± SEM of

determinations made in 4 to 6 separate cultures (15-20 cells assessed/culture).

Λρ<0.001 , compared to siRNA-Con. (B) Representative immunoblots of ER stress proteins and Ca²⁺ release channels in PC12 cells expressing VT, WT or A53T 24 h after transfection with siRNA-Con and siRNA-Herp (100 nM). Asterisk indicates the protein band corresponding to pan-RYR. The level of actin is not affected by siRNA treatment. (C) Histograms showing the viability of PC12 cells expressing VT, WT or A53T 24 h after transfection with siRNA-Con or siRNA-Herp (100 nM). Values represent the mean ± SEM of three independent experiments. #p<0.05; ^*p<0.01 , compared to the siRNA- Con treated groups. (D) Representative immunoblots of levels of ER stress proteins and Ca2+ release channels in PC12 cells expressing VT, WT or A53T after transfection with empty vector (-) or vector expressing Herp (+) for 48 h. Asterisk indicates the protein band corresponding to pan-RYR. (E-G) Representative immunoblots and results of densitometric analysis of the indicated protein levels in PC12 cells that were either transfected with the indicated siRNAs and collected 24 h after (Basal condition; left panels) or transfected with the siRNAs 8 h prior to incubation with tunicamycin (Tuni; 20 μg/ml)) for 24 h (ER stress condition; right panels). ^*p<0.01 , compared to the siRNA- Con treated groups. (H) qRT-PCR analysis of the relativeexpression of the indicated proteins in PC12 cells that were treated with vehicle control (Con), Tuni (20 μg/ml) or siRNA-Herp (100 nM) for 24 h. Values represent the mean ± SEM of three independent experiments. The mRNA level in Con was set at 1 . ^*p<0.01 , compared to Con.

FIG. 7: Herp interacts with and facilitates proteasomal-mediated degradation of ER-resident Ca²⁺ release channels (A) Representative immunoblots of the indicated ER Ca²⁺ release channels immunoprecipitated (IP) by anti-Herp antibody from lysates of PC12 that were treated with either tunicamycin (Tuni; 20 μς/ιηΙ) or vehicle for 16 h. The pre-immune normal IgG used as the negative control failed to yield an immunopositive band for IP3R or RYR. Input verifies the presence of these ER Ca²⁺ release channel protein in cell lysates. (B) Representative immunoblots of Herp, IP3R1 and pan-RYR protein levels in PC12 cells that were treated with the indicated doses of the proteasomal inhibitor MG- 132 for 3, 6, 12, and 24 h. (C) Representative immunoblots of Herp and IP3R1 protein levels in HEK293 cells that were transiently transfected with the indicated concentrations of an empty plasmid (Vector) or a plasmid expressing Herp for 24 and 48 h. pan-RYR was undetectable in HEK293 cells. (D) Representative immunoblots of Herp, IP3R, and pan- RYR protein levels in PC12 cells that were transfected with either an empty plasmid (Vector) or a plasmid expressing Herp 24 h prior to the addition of 1 μΜ MG-132. Cells were collected at the indicated time points after MG-132 addition.

FIG. 8: Elevation of ER stress markers and ER-resident Ca²⁺ release channels in A53T ccSyn mice

(A, B) Representative immunoblots(7\J and results of densitometric analysis (B) of the indicated ER stress proteins in lumbar spinal cords from age-matched non-transgenic (Non-Tg) and mutant aSyn (A53T) mice. A representative immunoblot confirming the expression of human aSyn in spinal cords of A53T is shown (upper panel). All immunoblots were reprobed for actin to control for equal protein loading (bottom panels). Values represent the mean ± SEM of four mice per group. ^*p<0.05; ^*p<0.01 , compared to Non-Tg mice. (C) Immunoprecipitation to quantify protein levels of IP3R1 (top) and pan-RYR (bottom) in lumbar spinal cords of 8 months-old Non-Tg and A53T mice. Each ER Ca²⁺ release channel protein was immunoprecipitated (IP) and immunoblotted (IB) with the respective antibodies. Pre-immune normal IgG was used as a negative control for IP. Histograms show the densitometric analysis of the band corresponding to each ER Ca²⁺ release channel protein. Values represent the mean ± SEM of four mice per group. ^*p< 0.01 compared to Non-Tg mice. (D) Representative immunoblots of IP3R1 (left) and pan-RYR (right) in protein complexes IP with anti-Herp antibody from lumbar spinal cord homogenates of 8 months-old Non-Tg and A53T mice. Pre-immune normal IgG was used as a negative control for IP. Asterisk denotes the specific band.

FIG. 9: Effects of tunicamycin and mutant aSyn on ER luminal Ca²⁺ levels, ER stress protein expression and cell survival.

(A) Representative recordings of the thapsigargin (Thap; 1 M)-induced elevation of intracellular Ca²⁺ ([ Ca²⁺ ]i) in PC12 cells expressing wild-type (WT) or mutant (A53T) a- Syn. PC12 cells transfected with empty vector (VT) were included as controls. Arrow indicates time of Thap addition. Cells were loaded with fura-2 and [Ca ]i was recorded in Ca²⁺ free medium as described under "Materials and Methods". Histograms show Ca²⁺ peak values (change from baseline) and AUC (area under the curve). Values are the mean ± SEM of determinations made in 4 to 6 separate cultures (15-20 cells assessed/culture). (B) Representative immunoblots (top) and results of densitometric analysis (bottom) showing time course of Herp, Grp94, Grp78 and CHOP protein levels in PC12 cells treated with either tunicamycin (Tuni; 20 μg/ml) (+) or vehicle alone (-). Values represent the mean ± SEM of three independent experiments. #p<0.05; ^*p< 0.01 , compared to vehicle. (C) Representative immunoblots (top) and results of densitometric analysis (bottom) of IP3R1 , pan-RYR, and PS1 protein levels in PC12 cells at the indicated time points after the addition of Tuni (+) or vehicle alone (-). Values represent the mean ± SEM of three independent experiments. #p<0.05; ^*p< 0.01 , compared to vehicle. (D) Histograms show the percent of viable cells after treatment of PC12-Tuni (top) and PC12-A53TaSyn (bottom) with the indicated doses of caffeine. PC12 cells were treated with caffeine either in vehicle for 2 or 24 h or in combination with Tuni for 26 h. In PC12 cells expressing VT, WT and A53T, caffeine was added 24 h after induction and then left incubated for another 24 h. Cell viability was determined by trypan blue exclusion. Values represent the mean ± SEM of three separate

experiments. ^*p<0.01 , #p<0.05, compared to vehicle control or VT at each time point and between the indicated groups. (E) qRT-PCR analysis of the relative expression of ER-stress proteins and Ca²⁺ release channels in PC12 cells treated with Tuni (+) for the indicated time points. Values represent the mean ± SEM of nine separate experiments. #p<0.05; ^*p< 0.01 , compared to vehicle. (F) qRT-PCR analysis of the relative

expression of IP3R1 and RYR1 in the indicated PC12 cells ectopically expressing empty vector (VT), wild-type (WT) or mutant (A53T) ccSyn. Cells were harvested 48 h after expression. Values represent the mean ± SEM of three separate experiments. #p<0.05; ^*p< 0.01 , compared to VT.

FIG. 10: Knockdown of ER Ca²⁺ release channel expression ameliorates ER stress (A, B) Representative immunoblots (top) and results of densitometric analysis (bottom) of Herp, Grp94/78, CHOP, and PS1 protein levels in PC12 cells that were transfected with siRNA-IP3R1 (250 nM) (A) or a combination of siRNA-RYR1 and siRNA-RYR3 (100 nM each) (B) for either 8 and 24 h prior to the addition of tunicamycin (Tuni; 20 μg/ml) or vehicle alone (Con). Values represent the mean ± SEM of three independent experiments. ^*p< 0.01 , compared to Con and between the indicated groups.

FIG. 11 : Inhibition of ER Ca²⁺ release reduces ccSyn inclusions formation (A) Representative images of aSyn inclusions (indicated by arrowhead) in PC12 cells transfected with either the GFP-tagged WTccSyn or GFP-tagged mutant (A53T) aSyn construct. Twenty four hours after transfection, PC12 cells were treated with

Xestospongin (5 μΜ), Salubrinal (Sal; 75 μΜ) or the respective vehicles for another 24 h, fixed, and counterstained with the nuclear dye 4',6-diamidino-2-phenylindole (DAPI; blue). (B) Histograms show the percentage of the indicated transfected PC12 cells with cytoplasmic aSyn protein inclusions. Values represent the mean ± SEM of three cultures in triplicate. #p< 0.05; ^*p< 0.01 , compared to vehicle controls.

FIG. 12: Levels of phospho-elF2a and total elF2a following tunicamycin and/or salubrinal treatments

A representative blot of phospho-elF2a and total elF2a in PC12 cells that were treated with either salubrinal (Sal; 75 μΜ) or tunicamycin (Tuni; 20 μg/ml) alone, or in

combination. Cells were harvested 12 h after treatments.

FIG. 13: Salubrinal reduces bradykin in-evoked Ca²⁺ transients in PC12-Tuni and PC12-aA53TSyn cells

(A) Representative images of the bradykinin-induced changes in the fluo-4 fluorescence intensity in PC12 cells that were treated with tunicamycin (Tuni; 20 μg/ml) for 18 h in the presence of salubrinal (Sal; 75 μΜ) or vehicle control (Con). (B) Representative recordings of the bradykinin (BK; 10 μM)-induced elevation of intracellular Ca²⁺ ([Ca²⁺ ]i) in PC12 cells after expression of mutant (A53T) a-Syn in the presence of Sal or Con. PC12 cells transfected with the empty vector (VT) were included as controls. Cells were loaded with fura-2 and [Ca²⁺]i was recorded in Ca²⁺ free medium as described under "Materials and Methods". Arrow indicates time of BK addition. Histograms show Ca²⁺ peak values (change from baseline) and AUC (area under the curve). Values are the mean ± SEM of determinations made in 4 to 6 separate cultures (15-20 cells

assessed/culture). #p<0.05,^*p<0.01 , ^Λρ<0.001 , compared to VT.

FIG. 14: Densitometric and qRT-PCR analyses of ER stress proteins and ER- resident Ca²⁺ release channel levels

(A, B) Results of densitometric analysis of ER stress proteins and ER Ca²⁺ release channels in the indicated PC12 cells 24 h after transfection with siRNA-Con or siRNA- Herp (100 nM). Values represent the mean ± SEM of three independent experiments. #p< 0.05; ^*p< 0.01 , compared between the indicated groups. (B) Results of

densitometric analysis of ER stress proteins and ER Ca²⁺ release channels in the indicated PC12 cells 48 h after ectopic expression of Herp. Values represent the mean ± SEM of three independent experiments. #p< 0.05; ^*p< 0.01 , compared between the indicated groups. (C, D) qRT-PCR analysis of the relative expression of ER-resident Ca²⁺ release channels in the indicated PC12 cells 24 h after addition of siRNAs or ectopic expression of Herp. Values are the mean ± SEM of three independent experiments. #p< 0.05; ^*p< 0.01 , compared between the indicated groups.

FIG. 15: Herp interacts with ER-resident Ca²⁺ release channels and A53TaSyn.

(A) A representative immunoblot shows absence of Grp78 in the protein complex immunoprecipitated (IP) by anti-Herp antibody from total lysates of PC12 cells treated with tunicamycin (Tuni; 20 μς/ιηΙ) or vehicle for 16 h. Input shows Grp78 in total lysates. The pre-immune normal IgG used as control for IP. (B) A representative immunoblot shows the presence of IP3R (top) and pan-RYR (bottom) in protein complexes IP by anti-c-Myc antibody from total lysates of HEK293 cells that were transiently transfected with the c-myc-tagged Herp for 24 h. The pre-immune normal IgG was used as the negative control for IP. Note that IP3R1 but not pan-RYR was readily detected in the inputs. (C) Representative immunofluorescence micrographs show co-localization of Herp (green) with IP3R (red) and pan-RYR (red) in PC12 cells that were treated with either vehicle (top) or Tuni (bottom) for 24 h. (D) Representative immunoblots show the presence of Herp or aSyn in protein complexes IP by anti-aSyn or anti-c-myc antibody, respectively, from total lysates of PC12 cells that were transiently transfected with both A53TaSyn and c-myc-tagged Herp for 24 h. The pre-immune normal IgG was used as the negative control for IP. Inputs verify the presence of c-myc-tagged Herp or aSyn in lysates. (E) Representative immunoblots show the presence of Herp or aSyn in protein complexes IP by anti-aSyn (top) or anti-Herp (bottom) antibody, respectively, from total lysates of PC12 cells that were transiently transfected with either Herp (top) or

A53TaSyn (bottom) for 24 h. The pre-immune normal IgG was used as the negative control for IP. Inputs verify the presence of c-myc-tagged Herp or aSyn in lysates.

FIG. 16: Blockade of proteasome inhibits Herp- induced degradation of ER- resident Ca²⁺ release channels

(A) Results of densitometric analysis of Herp, IP3R1 and pan-RYR protein levels in PC12 cells that were treated with the indicated doses of the proteasomal inhibitor MG- 132 or vehicle as control (Con) for 3, 6, 12, and 24 h. Values are the mean ± SEM of three independent experiments. #p<0.05; ^*p<0.01 , compared to Con. (B) Results of densitometric analysis of Herp, IP3R1 , and pan-RYR protein levels in PC12 cells transfected with either a plasmid expressing Herp or an empty plasmid (Vector) 24 h prior to the addition of 1 μΜ MG-132. Cells were collected at the indicated time points after MG-132 addition. Values are the mean ± SEM of three independent experiments. #p<0.05; ^*p<0.01 , compared between the indicated groups. FIG. 17: Herp interacts and co-localizes with the ubiquitin-interacting S5a subunit of the proteasome

(A) A representative immunoblot of Herp immunoprecipitated (IP) by anti-S5a antibody from total lyates of PC12 cells that were treated with Tuni (20 μg/ml) for 16 h. Pre- immune normal IgG was used as a negative control for IP. Inputs verify the amounts of Herp in lysates. Light chain indicates equal amounts of normal IgG and anti-S5a IgG in the protein complexes. (B) Representative immunofluorescence micrographs showing the co-localization of S5a (red) with either Herp or Grp78 (green) in PC12 cells exposed to Tuni or vehicle for 12 h. (C) A representative immunoblot of S5a protein in PC12 cells exposed to Tuni or vehicle for 16 h. (D) Representative immunofluorescence

micrographs showing the co-localization of S5a (red) with Herp (green) in PC12 cells transfected with siRNA-Con or siRNA-Herp 24 h prior to exposure to Tuni for another 14 h. FIG. 18: Accumulation of ER stress markers and of ER-resident Ca²⁺ channels in A53TaSyn transgenic mice

(A) Representative immunofluorescence micrographs showing the immunoreactivities of antibodies directed to the indicated proteins in the lumbar spinal cords from pre- symptomatic (5 months) and symptomatic (13-15 months) A53T mice. (B) mRNA levels of the indicated proteins in the lumbar spinal cords from 13-15 months old non- transgenic (Non-Tg) and mutant aSyn (A53T) mice. Values are the mean ± SEM of four mice per group. ^*p<0.01 , compared to Non-Tg. (C) A representative immunoblot of aSyn in protein complexes immunoprecipitated (IP) with anti-Herp antibody from lumbar spinal cord homogenates of 13-15 months old Non-Tg and A53T mice. Pre-immune normal IgG was used as a negative control for IP. Inputs verify the amounts of aSyn in spinal cord extract as determined by immunoblotting using an antibody to mouse and human aSyn.

DETAILED DESCRIPTION

Herp (Homocysteine-inducible ER stress protein) is an ER integral membrane protein with the amino-terminal ubiquitin-like domain projecting into the cytosol (14). Upregulation of Herp is essential for neuronal survival as Herp knockdown enhances vulnerability to ER stress-induced apoptosis (15, 16). How Herp contributes to the restoration of ER homeostasis is unclear. Herp appears to stabilize ER Ca²⁺

homeostasis and mitochondrial function in neural cells subjected to ER stress (16). Herp may also play an essential role in ER-associated protein degradation (ERAD), the primary mechanism of misfolded protein degradation, as its knockdown results in the selective accumulation of ERAD substrates (17). Recent studies demonstrated that Herp is induced in PD substantia nigra and is present in the core of Lewy bodies (18). The roles of Herp in PD remain unknown. Because Herp was shown to be critical for survival adaptation in the neurotoxin models of PD (19), disclosed herein are studies showing that Herp may counteract the neurodegenerative cascades caused by induced expression of mutant aSyn. It has been found out that Herp plays an essential role in suppressing mutant aSyn-induced activation of ER stress-associated apoptosis signaling by inhibiting the deregulated ER Ca²⁺release associated with the aberrant accumulation of ER resident Ca²⁺ release channels.

Certain embodiments described herein, relate, in part, to the discovery that deregulation of intracellular Ca²⁺ homeostasis actively participates in neuronal death associated with ER stress, which in turn relates to the pathogenesis of PD and several other neurodegenerative diseases such as Alzheimer's, Huntington's and prion diseases . It has been discovered that inhibiting elF2a phosphatase in neural tissue assists in maintaining Ca²⁺ homeostasis, even in cells having elevated levels of aSyn.

According to another embodiment, disclosed herein is a method of stabilizing Ca²⁺ homeostasis in affected neurons of a subject. The method involves administering a therapeutically effective amount of an elF2a phosphatase inhibitor to the subject. In a specific embodiment, the method involves administering a therapeutically effective amount of salubrinal.

According to another embodiment, provided herein is a method of treating or delaying the onset of a neurodegenerative disease associated with deregulation of intracellular Ca²⁺ homeostasis in a subject. The method involves administering to the subject a therapeutically effective amount of an elF2a phosphatase inhibitor.ln a specific embodiment, the method involves administering a therapeutically effective amount of salubrinal.

In certain embodiments described herein, the subject being treated with elF2a phosphatase inhibitor has exhibited one or more symptoms of a neurodegenerative disease. Examples of motor symptoms include resting tremor, bradykinesia, muscle rigidity, postural instability, freezing of gait, micrographia, "mask-face", or uncontrolled accelerative movements. Examples of nonmotor symptoms include memory

impairment, disorientation, misinterpreting spatial relationships, personality changes; or impairment of familiar tasks. Examples of other motor symptoms include twitching and cramping of the muscles, especially those in the hands and feet, muscle weakness in the arms or legs, loss of motor control in the arms or legs, general weakness and fatigue, tripping and falling, dropping things, impaired speech, or difficulty chewing or swallowing.

According to another embodiment, disclosed herein is a method of treating or delaying the onset, or preventing symptoms of a neurodegenerative disease in a subject in need thereof. The method involves ameliorating an aberrant accumulation of ER- resident Ca²⁺ release channels in neurons of the subject. In a specific embodiment, the amelioration of such channels is accomplished by administering a therapeutically effective amount of salubrinal.

"Neurodegenerative disease," as used herein, refers to any condition

characterized by the progressive loss of neurons, due to cell death, in the central nervous system of a subject. Examples of neurodegenerative diseases include, but are not limited to, Alexander disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt- Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,

Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe disease, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Neuroborreliosis, Parkinson's disease, Pelizaeus- Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases,

Refsum's disease, Sandhoff disease, Schilder's disease, Sub-Acute Combined

Degeneration of the Cord Secondary to Pernicious Anaemia, Schizophrenia,

Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease),

Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis and Charcot-Marie- Tooth disease. In certain specific embodiments, disclosed herein are methods for treating Parkinson's disease, Alzheimer's disease, or Amyotrophic lateral sclerosis.

"Subject", as used herein, means an animal including a human or non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate, or an insect, such as a fly.

"Treat," "treatment," and "treating," as used herein refer to any of the following: the reduction in severity of a disease or condition; delaying the progression of the disease; the amelioration of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition; the prophylaxis of one or more symptoms associated with a disease or condition; restoring calcium homeostasis is cells, particularly neurons; controlling aberrant accumulation of ER-resident calcium channels in neurons; or interfering with alpha-syn association with HERP.

"Therapeutically effective amount" as used herein refers to a dose or series of doses over time that is effective for treatment.

"Alzheimer's disease," (AD) as used herein, refers to a progressive degenerative disease of the brain that causes impairment of memory and dementia manifested by confusion, visual-spatial disorientation, inability to calculate and deterioration of judgment. Atrophy of the cerebral cortex, with consequent enlargement of sulci and ventricles may be grossly evident on imaging studies. Histologically the cortex, hippocampus and amygdala show atrophy of neurons, with cytoplasmic vacuoles and argentophillic granules; distortion of intracellular neurofibrils (neurofibrillary tangles) due to excessive phosphorylation of microtubular tau proteins; and plaques composed of granular or filamentous argentophillic masses with a core of the 42 amino acid form of β amyloid (Αβ ₄₂ ). The concentration of tau protein in the cerebrospinal fluid is increased, while the concentration of Αβ ₄₂ is decreased.

"Amyotrophic lateral sclerosis (ALS)" is a fatal neurodegenerative disease that affects selectively the motoneurons in the central nervous system. Most ALS patients die within five years of onset, and the mechanisms of the onset of the disease, as well as of its progression are poorly understood.

Pharmaceutical Compositions

Aspects of the present invention also provide pharmaceutical compositions comprising one or more of the compounds described. The pharmaceutical compositions can be administered to a patient to achieve a desired therapeutic effect, e.g., inhibiting elF2a phosphatase activity. The compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a subject alone, or in combination with other therapeutic agents or treatments as described below.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the subject. In certain embodiments, the compositions are formulated for topical administration, such as in the form of a cream or gel. The cream or gel can be formulated as an aqueous fluid containing a soluble polymer as the thickening agent, for example. Alternately, the cream or gel may comprise a suspension or a colloidal solution, which contains insoluble particles suspended in a liquid carrier medium. See U.S. Patent No. 5,208,031 , the entirety of which is hereby incorporated by reference. Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, Pa., which is incorporated herein by reference). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose for any one or more of the compounds described herein is within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which provides the desired result. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

In a specific embodiment, normal dosage amounts can vary from 0.1 - 1 .5 mg/kg depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.

Conjunctive Therapeutic Agents

In any of the embodiments described above, any of the compounds and/or compositions of the invention can be co-administered with other appropriate therapeutic agents (conjunctive agent or conjunctive therapeutic agent) or therapies for the treatment or prevention of a neurodegenerative disorder, and/or symptom(s) thereof. Selection of the appropriate conjunctive agents or therapies for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents or therapies can act synergistically to effect the treatment or prevention of the diseases or a symptom thereof. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

In certain embodiments, the conjunctive agents may be an agent for treating PD or AD as is known in the art. Exemplary agents for PD include, but are not limited to, dopamine-type drugs such as levodopa/carbidopa, dopamine agonists, anticholinergics, eldepryl, deprenyl, COMT inhibitors. Exemplary agents for AD include, but are not limited to, cholinesterase inhibitors, memantine, or vitamin E.

The mode of administration for a conjunctive formulation in accordance with the present invention is not particularly limited, provided that the compounds of the present invention as described herein ("elF2a phosphatase inhibitor") and the conjunctive agent are combined upon administration. Such an administration mode may, for example, be (1 ) an administration of a single formulation obtained by formulating a elF2a phosphatase inhibitor and a conjunctive agent simultaneously; (2) a simultaneous administration via an identical route of the two agents obtained by formulating a novel elF2a phosphatase inhibitor and a conjunctive agent separately; (3) a sequential and intermittent administration via an identical route of the two agents obtained by formulating a elF2a phosphatase inhibitor and a conjunctive agent separately; (4) a simultaneous administration via different routes of two formulations obtained by formulating a elF2a phosphatase inhibitor and a conjunctive agent separately; and/or (5) a sequential and intermittent administration via different routes of two formulations obtained by formulating a elF2a phosphatase inhibitor and a conjunctive agent separately (for example, a elF2a phosphatase inhibitor followed by a conjunctive agent, or inverse order) and the like.

The dose of a conjunctive formulation may vary depending on the formulation of the elF2a phosphatase inhibitor and/or the conjunctive agent, the subject's age, body weight, condition, and the dosage form as well as administration mode and duration. One skilled in the art would readily appreciate that the dose may vary depending on various factors as described above, and a less amount may sometimes be sufficient and an excessive amount should sometimes be required.

The conjunctive agent may be employed in any amount within the range causing no problematic side effects. The daily dose of a conjunctive agent is not limited particularly and may vary depending on the severity of the disease, the subject's age, sex, body weight and susceptibility as well as time and interval of the administration and the characteristics, preparation, type and active ingredient of the pharmaceutical formulation. An exemplary daily oral dose per kg body weight in a subject, e.g., a mammal, is about 0.001 to 2000 mg, preferably about 0.01 to 500 mg, more preferably about 0.1 to about 100 mg as medicaments, which is given usually in 1 to 4 portions.

When the elF2a phosphatase inhibitor and a conjunctive agent are administered to a subject, the agents may be administered at the same time, but it is also possible that a conjunctive agent is first administered and then a elF2a phosphatase inhibitor is administered, or that a elF2a phosphatase inhibitor is first administered and then a conjunctive agent is administered. When such an intermittent administration is employed, the time interval may vary depending on the active ingredient administered, the dosage form and the administration mode, and for example, when a conjunctive agent is first administered, a elF2a phosphatase inhibitor may be administered within 1 minute to 3 days, preferably 10 minutes to 1 day, more preferably 15 minutes to 1 hour after the administration of the conjunctive agent. When a elF2a phosphatase inhibitor is first administered, for example, then a conjunctive agent may be administered within 1 minute to 1 day, preferably 10 minutes to 6 hours, more preferably 15 minutes to 1 hour after the administration of a elF2a phosphatase inhibitor.

Examples Example 1 : Expression of Mutant aSyn Evokes a Sustained ER Stress Response

Previous studies provide evidence that mutant aSyn triggers a cell death program that involves activation of the ER stress response (1 1 ). It is yet not clear which and how ER stress proteins contribute to mutant aSyn-induced cell death. To

investigate the role of Herp in the mutant aSyn-induced degenerative process, Tet- inducible PC12 cells were generated. Time course analysis indicated that aSyn protein level reaches a plateau 48 h after induction (FIG. 1A). Concurrently, mRNA and protein levels of the ER stress markers Grp78 and Herp were markedly elevated in the PC12 cells expressing mutant aSyn, especially those expressing A53TaSyn (PC12-A53T aSyn), when compared to PC12 cells expressing wild-type aSyn (PC12-WTaSyn)(FIG. 1 B). Levels of CHOP were also markedly higher in PC12-A53T aSyn (FIG. 1 B)

suggesting, that at this expression level, there was a selective deleterious effect of A53TaSyn but not WT aSyn. Similar results were obtained in PC12 cells transiently expressing A53TaSyn and WTaSyn (FIG. 1 C). Example 2: Herp Protects Against Mutant aSyn-lnduced Cell Death

Compared to PC12 cells stably expressing the empty vector (PC12-VT), PC12- WTaSyn and PC12-A30P aSyn, PC12-A53T aSyn exhibits significantly higher baseline cell death (FIG. 2A) which correlated with increased CHOP protein level and caspase- 12 activation (FIG. 2B). Because A53T aSyn enhances activation of ER stress-related apoptosis signaling, PC12-A53TaSyn was utilized in subsequent knockdown studies. PC12-A53TaSyn treated with a small interference RNA (siRNA) targeting Herp (siRNA- Herp) but not a non-silencing control siRNA (siRNA-Con) exhibited higher basal rate of cell death (FIG. 2C). In contrast, ectopic expression of Herp, but not the dominant- negative mutant AUBL-Herp that lacks the UBL domain (FIG. 2D), significantly improved the viability of PC12-A53TaSyn (FIG. 2E). Notably, AUBL-Herp appeared to potentiate A53TaSyn -induced cell death consistent with a dominant-negative action of AUBL-Herp as reported previously (19).

Example 3: Mutant aSynPerturbs ER Ca²⁺ Homeostasis During ER Stress

Given that Herp protects from A53TaSyn-induced death (FIG2C, 2E) and that Herp plays a crucial role in stabilizing ER Ca²⁺ homeostasis in ER-stressed PC12 cells (16), it was determined whether A53TaSyn may perturb ER Ca²⁺ regulation by altering the activity of the two main classes of ER-resident Ca²⁺ release channels, IP3R (inositol triphosphate receptor) and RYR (ryanodine receptor) which can be activated by their respective agonists, bradykinin and caffeine (20-22). The average peak amplitude of bradykinin-evoked Ca²⁺ release in the absence of extracellular Ca²⁺ was significantly larger in PC12-A53T aSyn when compared to PC12-WTaSyn and PC12-VT (FIG. 3A) indicating that A53TaSyn enhances ER Ca²⁺ release. No significant difference was observed in thapsigargin-induced depletion of ER Ca²⁺ store (FIG. 9A) suggesting that the ER stress-induced perturbation of intracellular Ca²⁺ level ([Ca²⁺ ]i) in PC12- A53TaSyn cannot be explained by higher ER luminal Ca²⁺ but rather is caused by a higher fraction of ER Ca²⁺ being released via IP3R.

Tunicamycin (Tuni) is a classical ER stressor that induces a sustained increase of ER stress proteins (FIG. 9C). The magnitude of the bradykinin-evoked Ca²⁺ release was also higher in PC12 cells treated with Tuni (PC12-Tuni) when compared to control cells that were left untreated or treated with vehicle (data not shown; 16,19).

Consequently, treatment with BAPTA-AM, a cell permeable Ca²⁺ chelator, markedly improves the viability of both PC12-A53TaSyn and PC12-Tuni (FIG. 3B) suggesting that Tuni and A53TaSyn increase susceptibility to ER stress-induced death by enhancing ER Ca²⁺ release.

Example 4: Mutant aSyn-lnduced ER Stress Perturbs Homeostatic Regulation of ER-Resident Ca²⁺ Release Channels

Next, it was determined whether the heightened cytosolic Ca²⁺ level in PC12- Tuni and PC12-A53TaSyn results from altered homeostatic regulation of ER-resident Ca²⁺ release channels. Three distinct types of IP3Rs (types 1-3) have been cloned in mammals and each type shows distinct properties in terms of their IP3 sensitivity, modulation by cytoplasmic Ca²⁺ concentration, and unique tissue distribution (23,24). Among them, the type 1 IP3R (IP3R1 ) is highly expressed in the central nervous system (24). qRT-PCR analysis showed that IP3R1 is the major IP3R isoform expressed in PC12 cells (unpublished data). PC12 cells also express RYR1 and RYR3 (pan-RyR) (20, 21 ). Levels of IP3R2, IP3R3, and RYR2 mRNAs were not assessed due to their low abundance in PC12 cells. PC12-Tuni exhibit marked accumulation of IP3R1 and pan- RyR protein (FIG. 3D; FIG. 9C) consistent with the notion that Tuni-induced ER stress leads to disruption of ER Ca homeostasis (16). Expression of A53TaSyn also induces a marked increase in the protein levels of IP3R1 (FIG. 3E) and pan-RyR (FIG. 3F) suggesting that the aberrant accumulation of ER Ca²⁺ release channels was likely mediated through a common ER stress-related mechanism. Consistent with the elevated pan-RYR protein levels, PC12-Tuni and PC12-A53TaSyn were more

vulnerable to cell death in the presence of caffeine when compared to their respective controls (FIG. 9D). By contrast, level of presenilis (PS1 ) which functions as a passive ER Ca²⁺ leak channel (25), was not markedly altered by ER stress (FIG 3D, E). It is worth noting that unlike the increase of ER stress proteins which is mediated by a transcriptional mechanism (6, 7), the ER stress-associated accumulation of IP3R1 and pan-RYR was independent of transcription (FIG. 9E, F).

Example 5: Inhibition of Deregulated ER Ca²⁺ Release Ameliorates ER Stress- Mediated Cell Death and aSyn Aggregation

Because ER-released cytosolic Ca²⁺ plays a critical role in the activation of several death effector pathways (16), it was next determined whether blockade of ER Ca²⁺ release may ameliorate ER stress-induced cell death. Xestospongin C (a blocker of IP3R) and dantrolene (a RyR blocker) at doses that did not cause robust death within 24 h substantially improved the viability of PC12-Tuni (FIG 4A) and PC12-A53TaSyn (FIG. 4B). Neither IP3R nor RyR inhibition altered the expression of aSyn (data not shown), thereby confirming that inhibition of ER Ca²⁺ release rather than reduced expression of A53TaSyn contributes to cell protection.

To further determine whether these ER-resident Ca²⁺ release channels are responsible for the heightened sensitivity of PC12-A53TaSyn to ER stress-mediated cell death, each channel protein was knocked down at a time by using either siRNA-IP3R1 or siRNA-RYR1 and siRNA-RYR3 in combination (FIG. 4C). The non-silencing control siRNA (siRNA-Con) alone did not alter IP3R1 nor RYR1 /RYR3 expression (not shown). A close correlation between protein levels of these ER Ca²⁺ release channels and the ER stress-induced apoptotic mediator CHOP (FIG. 10) was observed in PC12-Tuni (FIG. 4D) and PC12-A53TaSyn (FIG. 4E). CHOP which is known to be upregulated following a severe or prolonged ER stress, was markedly suppressed along with Herp and Grp94/78 in PC12-Tuni transfected with the silencing siRNAs (FIG. 10). These data suggest that impaired homeostatic regulation of ER-resident Ca²⁺ release channels might underlie chronic activation of ER stress and associated apoptosis signaling. Because ER-released cytosolic Ca²⁺ plays a role in promoting aSyn aggregation

(26), we next examined aSyn inclusion formation in PC12 cells transiently transfected with either WTaSyn or A53TaSyn tagged to green fluorescent protein (GFP) by fluorescence microscopy. Xestospongin C substantially reduces not only the fraction of cells bearing cytoplasmic aSyn inclusions but also the size of the inclusions (FIG. 11). These fluorescent aggregates were not detected in PC12 cells transfected with GFP alone (data not shown).

Example 6: Salubrinal Inhibits ER Stress-mediated Cell Death by Preventing the Aberrant Accumulation of ER-Resident Ca²⁺ Release Channels

Next, the question was asked whether salubrinal, a compound that has been shown to ameliorate A53TaSyn-induced cell death (1 1 ), may counteract prolonged ER stress through the homeostatic regulation of ER-resident Ca²⁺ release channels.

Salubrinal at a dose that inhibits the cellular phosphatase complexes that

dephosphorylate eiF2a (FIG. 12) not only blocks the ER stress-associated increase of IP3R and pan-RYR but also dramatically reduces protein levels of Herp, Grp94/78 and CHOP in both PC12-Tuni (FIG. 5A) and PC12- A53TaSyn (FIG. 5B) suggesting that this compound likely ameliorates ER stress by improving the homeostatic regulation of ER Ca²⁺ release channels through a mechanism that is independent of transcription (FIG. 5C). By contrast, salubrinal did not alter PS1 protein level in PC12-Tuni (FIG. 5A) and PC12-A53TaSyn (FIG. 5B). The salubrinal-mediated decrease of ER Ca²⁺ release channels was accompanied by a substantial reduction in the bradykinin-evoked Ca²⁺ transients in both PC12-Tuni (FIG. 13A) and PC12-aA53TSyn (FIG. 13B).

Consequently, salubrinal also significantly ameliorates the Ca²⁺ dependent aggregation of A53TaSyn-GFP in the cytosol (FIG. 11). Example 7: Herp Counteracts ER Stress Through the Homeostatic Regulation of ER-Resident Ca²⁺ Release Channels

Because Herp counteracts Tuni-induced cell death through the stabilization of ER Ca2+ homeostasis (16), it was determined whether Herp protects PC12-A53TaSyn (FIG. 2C, D) by a similar mechanism. Knockdown of Herp substantially increases the amplitude of the bradykinin (BK)- induced Ca2+ transients (FIG. 6A) that result from the marked accumulation of IP3R1 (FIG. 6B; FIG. 14A). Levels of pan-RYR but not PS1 proteins were also affected by Herp knockdown (FIG. 6B; FIG. 14). Consequently, the deficits in Herp-dependent homeostatic regulation of ER Ca²⁺ release channels is also accompanied by increased levels of the ER stress markers Grp94/78 and CHOP (FIG. 6B; FIG. 14A) and enhanced vulnerability to aSyn-induced death (FIG. 6C).

Conversely, ectopic expression of Herp suppresses the aberrant accumulation of IP3R1 and pan-RYR but not PS1 proteins (FIG. 6D; FIG. 14B). Neither knockdown nor ectopic expression of Herp alters imRNA levels of IP3R1 and pan-RYR (FIG. 14C, D)

suggesting that, analogously to salubrinal, Herp promotes the homeostatic regulation of these ER-resident Ca²⁺ release channels through a mechanism that is independent of transcription.

Knockdown of Herp also increases basal (FIG. 6E, F) and stress-induced accumulation (FIG. 6E, G) of both IP3R1 and pan-RYR proteins independently of transcription (FIG. 6H) in PC12-Tuni. Consistent with the notion that Herp counteracts Tuni-induced death (19), knockdown of Herp results in a significant increase of CHOP protein by transcriptional regulation (FIG. 6 E, H).

Example 8: Herp Promotes Degradation of ER-Resident Ca²⁺ Release Channels Through ERAD. Because Herp has been shown to bind to and target protein substrates for ERAD

(17), we next tested whether Herp modulates the levels of IP3R1 and/or pan-RYR proteins by a similar mechanism. Immunoprecipitation with an anti-Herp antibody followed by immunoblotting with antibodies to each ER Ca²⁺ release channel demonstrated that a greater fraction of Herp forms a complex with IP3R1 and pan-RYR in PC12-Tuni when compared to vehicle-treated control cells (FIG. 7A). The specificity of the interaction was confirmed by immunoblotting the Herp-containing protein complex with an antibody to Grp78 (FIG. 15A) and by performing the co-immunoprecipitation assay using lysates from HEK293 expressing c-myc-tagged Herp (FIG. 15B). Neither the pre-immune normal IgG nor Grp78 antibody forms a protein complex with Herp. Double immunofluorescence labeling confirmed Herp colocalization with each ER Ca²⁺ release channel protein in PC12-Tuni (FIG. 15C). Herp also interacts with A53TaSyn (FIG. 15D, E) suggesting that this interaction could possibly interfere with the protective role of Herp (see discussion).

To determine whether binding of Herp to IP3R1 and pan-RYR results in proteasome-mediated protein degradation of these Ca²⁺ release channel proteins, PC12 cells were treated with the proteasome inhibitor MG-132. Consistent with the notion that the degradation of IP3R1 and pan-RYR proteins is mediated by the proteasomes (27, 28), MG-132 markedly increases steady-state protein levels of these ER-resident Ca²⁺ release channels (FIG. 7B; FIG. 16A). Ectopic expression of Herp results in a significant reduction of IP3R1 and pan-RYR protein levels (FIG. 7C) that can be reversed upon inhibition of proteasome activity (FIG. 7D, FIG. 16B). Note that MG- 132 also increases Herp protein levels (FIG. 7B, D) suggesting that Herp itself is a proteasome substrate (29). In support for this notion, Herp interacts and co-localizes with the ubiquitin-interacting S5a subunit of the proteasome in PC12-Tuni (FIG. 17A). Increased co-localization of S5a with the Grp78-labeled ER was also detected in PC12 cells transfected with Herp (FIG. 17B). Though S5a protein level was not markedly altered in PC12-Tuni (FIG. 17C), knockdown of Herp substantially reduces S5a co- localization with the ER in PC12-Tuni suggesting that ER stress-induced upregulation of Herp but not S5a is sufficient for the recruitment of proteasomes to the ER (FIG. 17D). Collectively, our data indicate that aberrant accumulation of IP3R and pan-RYR perturbs ER Ca²⁺ homeostasis in ER stressed cells and that Herp prevents aberrant ER Ca²⁺ release by targeting these ER-resident Ca²⁺ release channels for ERAD. Example 9: Accumulation of ER Stress Markers and ER-Resident Ca²⁺ Channels in A53TaSyn Transgenic Mice

Next, it was explored whether A53TaSyn-induced ER stress markers and ER Ca2+ channels were detected in vivo. Transgenic mice overexpressing A53TaSyn (A53T mice) develop motor abnormalities associated with the accumulation of aSyn inclusions in spinal cord motor neurons (30). Immunoblotting reveals marked

upregulation of Herp and Grp78/Bip proteins in spinal cords of >8 months old A53T mice (symptomatic) when compared to 2 months old A53T mice (pre-symptomatic) and non-transgenic (Non-Tg) mice (FIG. 8A, B). CHOP protein was low in Non-Tg mice but was readily detected in A53T mice (FIG. 8A, B). Immunohistochemistry indicates a marked increase of nitrated aSyn in NeuN-labeled spinal cord neurons and further confirms the increase of ER stress markers and ER-resident Ca²⁺ release channels in 13-15 months old (symptomatic) when compared to 5 months old (pre-symptomatic) A53T mice (FIG. 18A). By contrast to the ER stress markers, the upregulation of ER- resident Ca²⁺ release channels was not attributed to increased expression (FIG. 18B). The amounts of IP3R1 and pan-RYR in the spinal cord homogenates that form a protein complex with Herp were also markedly higher in A53T compared to Non-Tg mice (FIG. 8C) consistent with the notion that ERAD may contribute to the homeostatic regulation of ER Ca²⁺ release channel proteins in spinal cord motor neurons. The interaction between Herp and A53TaSyn was also confirmed in spinal cords of symptomatic transgenic mice (FIG. 18C) suggesting that this interaction may impair the ability of Herp to prevent the aberrant accumulation of ER-resident Ca²⁺ release channels and, hence, its ER Ca²⁺ -stabilizing action in ER stressed motor neurons. These findings link aberrant ER Ca²⁺ regulation and chronic ER stress to motor neuron dysfunction and death in the pathophysiology of synucleinopathies.

Discussion of results provided in Examples 1-9

Neuronal loss in both familial and sporadic forms of neurodegenerative disorders is accompanied by formation of protein inclusions or fibrillar aggregates composed of misfolded proteins that can induce ER stress. The accumulation of evidence that ER stress is critically involved in the pathogenesis of neurodegenerative disorders suggests that approaches that aim to halt ER stress may prevent the pathological cascades induced by protein inclusions. There is growing evidence that the ER can play pivotal roles in regulating cell survival and apoptosis in a variety of cell types including neurons (30, 31 ), but the mechanisms linking ER stress to apoptosis are not understood. The identification of conditions that slow ER stress may reveal novel strategies for counteracting ER stress-mediated cell death.

The ER is the major intracellular store of Ca²⁺ and aberrant regulation of luminal ER Ca²⁺ is thought to play critical roles in many apoptotic cascades (31 ). Deregulated ER Ca²⁺ homeostasis has also been implicated in the pathophysiology of chronic neurodegenerative diseases such as prion disorders, Huntington's and Alzheimer's (32- 34). Here it is shown that A53TaSyn evokes ER stress and that the attendant disturbances in ER Ca²⁺ homeostasis contributes to a higher sensitivity to ER stress- induced cell death. It is demonstrated herein that Herp counteracts A53TaSyn-induced cell death by stabilizing ER Ca²⁺ homeostasis. Ectopic expression of Herp markedly reduced A53TaSyn-induced toxicity whereas knockdown of Herp exacerbates or prolongs ER stress leading to a significant augmentation of toxicity. Hence, a better understanding of the function of Herp is therefore of high significance to elucidate the functional link between the ER stress and ER Ca²⁺ homeostasis and to develop mechanism-based neuroprotective strategies for PD and related neurodegenerative diseases.

The underlying molecular mechanism(s) whereby Herp modulates ER Ca²⁺ homeostasis remains poorly understood. Knockdown of Herp leads to the accumulation of IP3R1 and pan-RyR proteins in PC12 cells and, consequently, promotes aberrant ER Ca2+ release that in turn may decrease the threshold for the activation of ER stress- related cell death pathways. Consistent with this notion, gene knockdown and pharmacological inhibition of ER Ca²⁺ release channels ameliorates ER stress and protects PC12- A53TaSyn and PC12-Tuni against ER stress-induced cell death (Figure 2- 4). Conversely, overexpression of Herp stabilizes ER Ca²⁺ homeostasis and inhibits ER stress-induced cell death by preventing the accumulation of ER Ca Ca²⁺ release channel proteins in PC12-A53TaSyn. It is noteworthy that the accumulation of IP3R1 and pan-RyR proteins was partially suppressed in spite of the elevated level of endogenous Herp in PC12-A53TaSyn suggesting that binding of Herp to A53TaSyn (Supplementary Figure 2- 7D) and its accumulation in the core of Lewy bodies (18) may interfere with its protective function and that ectopically expressed Herp can overcome this A53TaSyn-mediated inhibition.

Mechanistically, Herp interacts with and facilitates the degradation of ER Ca²⁺ release channel proteins by ERAD. Several recent studies support a role for Herp in ERAD (17) based on the notion that Herp is rapidly degraded in a proteasome- dependent fashion (29) and that knockdown of Herp leads to the accumulation of several established ERAD substrates (17). Herp has been shown to interact with Hrdl p, a membrane-anchored E3 ligase that is required for ERAD (17), and with ubiquilin, a shuttle protein that delivers ubiquitinated substrates to the proteasome for degradation (35). It was found that Herp knockdown in ER stressed cells leads to the accumulation of both IP3R1 and pan-RYR. Conversely, ectopic expression of Herp prevents the accumulation of these ER Ca²⁺ release channels. Treatment with MG-132 not only elevates the basal level of IP3R1 and pan-RYR proteins but also prevents the ability of Herp to inhibit their accumulation in ER stressed cells (Figure 2- 7D) suggesting the critical involvement of ERAD in the homeostatic regulation of these ER Ca²⁺ release channels. Deletion and function analyses further support the involvement of ERAD in Herp-mediated cell protection via the stabilization of ER Ca²⁺ homeostasis. Ectopic expression of Herp lacking the UBL-domain which functions as a proteasome- interacting domain (17, 43) fails not only to stabilize ER Ca²⁺ homeostasis but also to protect PC12-Tuni (16) and PC12-A53TaSyn from ER stress-induced death (Figure 2- 2E). Notably, salubrinal appears to modulate the vulnerability of PC12 cells to ER stress-induced cell death by preventing the accumulation of ER Ca²⁺ channels (Figure 2- 6).

The data presented herein provides the first evidence that ER stress is regulated by the activity of ER-resident Ca²⁺ release channels. It was found that pharmacological inhibition or knockdown of ER Ca²⁺release proteins ameliorates ER stress-induced cell death suggesting that aberrant ER Ca²⁺ release is associated with higher susceptibility to chronic enhancement of ER stress. Though the detailed mechanisms underlying Ca²⁺ dependent cell death in PC12-A53TaSyn was not investigated in the present study, it is likely that accumulation of ER Ca²⁺ release channels leads to enhanced ER to mitochondria Ca²⁺flow that triggers the loss of mitochondrial membrane potential and increased generation of reactive oxygen species (ROS) (16). Previous studies demonstrate that ROS-induced damage to the ER may amplify Ca²⁺ release via a mechanism involving oxidation-induced activation of RYR and IP3R (36). Ectopic expression of Herp has been shown to counteract this deleterious positive feedback loop by inhibiting the proapoptotic Ca²⁺ flow from the ER to mitochondria in PC12 cells exposed to the PD-inducing toxin 1 -methyl-4-phenylpyridinium (MPP+) (19). The increase of CHOP detected in PC12-Tuni, PC12-A53TaSyn and siRNA-Herp treated 60 PC12 cells likely results from the depletion of ER Ca²⁺ store associated with the aberrant accumulation of IP3R1 and pan-RYR as ectopic expression of Herp

counteracts CHOP upregulation by promoting the homeostatic regulation of these ER Ca²⁺ channel proteins.

It is noteworthy that chronic enhancement of ER stress resulting from the disruption of ER Ca²⁺ homeostasis could trigger aSyn protein aggregation in the cytosol and that blockade of ER Ca²⁺release channels (Suppl Figure 2- 2) ameliorates aSyn inclusion formation suggesting a causative link between chronic ER stress and aSyn oligomer formation. Consistent with this notion, sustained ER Ca²⁺ release triggered by thapsigargin accelerates the formation of potentially cytotoxic oligomers in aSyn-GFP transfected cells (26). Tuni at doses that induce chronic stress associated with sustained ER Ca²⁺release (16, 37) has also been shown to promote the accumulation of aSyn oligomers (38). Because Sal ameliorates ER stress and protects PC12- Tuni and PC12-A53TaSyn, it is believed, without being bound to any particular theory, that its neuroprotective action may be due to improved regulation of ER Ca²⁺ homeostasis. In support of this notion, Sal inhibits the aberrant accumulation of ER-resident Ca²⁺release channels (Figure 2-5) and prevents aSyn aggregation (Suppl Figure 2- 5). Consistent with the findings in PC12-A53T cells, higher levels of several ER stress markers including the ER stress-induced apoptotic mediator CHOP, and ER- resident Ca²⁺release channels in the spinal cords of symptomatic A53T mice were detected when compared to Non-Tg and pre-symptomatic A53T mice suggesting that accumulation of A53TaSyn promotes motor neuron degeneration in part by a

mechanism involving chronic ER stress associated with the deregulation of ER

Ca²⁺homeostasis. In addition to the elevation of Herp protein, increased interaction of Herp with A53TaSyn in spinal cord homogenates of symptomatic A53T mice was detected, which further supports the notion that Herp-dependent ERAD of ER-resident Ca²⁺release channels may be impaired in vulnerable motor neurons.

Dopaminergic neurons appear to be relatively resistant to degeneration in A53T mice (30, 39) and express relatively high levels of the Ca²⁺-binding protein calbindin (39). By contrast, spinal cord motor neurons are characterized by low cytosolic

Ca²⁺buffering capacities (40) and, hence, may be more susceptible to chronic ER stress induced by A53TaSyn and associated degenerative processes triggered by the aberrant ER Ca²⁺release. Future studies will determine whether direct modulation of Herp expression in vivo may impact the levels of ER-resident Ca²⁺ release channel proteins, aSyn inclusion formation, disease manifestations and progression. Because ER stress elicited by the aggregation of amyotrophic lateral sclerosis-linked mutant superoxide dismutase 1 (SOD1 ) has been implicated in motor neuron death (41 ) and because salubrinal delays the disease process and extends the lifespan of mutant G93A-SOD1 mice (42), elucidation of the cellular and molecular mechanisms that promote or prevent disturbances in ER Ca²⁺ homeostasis will likely lead to novel approaches for therapeutic intervention for synucleinopathies and motor neuron diseases. Materials and Methods related to Examples 1-9

Cells, Plasmid and Reagents

Pheochromocytoma 12 (PC12) and human embryonic kidney 293 (HEK293) cells were purchased from ATTC. PC12 cells were selected because they are dopaminergic and have been extensively studied as models of neuronal degeneration. The pcDNA3.1 plasmids containing the c-myc-tagged full-length or loss-of-function deletion of human Herp cDNA have been described previously (16, 19). Xestospongin C (Tocris), dantrolene (Sigma), bradykinin (Sigma) were prepared as concentrated 1000x stocks in dimethylsulfoxide (DMSO; Sigma) or Lock's solution (mM): NaCI, 154; KCI, 5.6; CaCI2, 2.3; MgCI2, 1 .0; NaHCO3, 3.6; glucose, 10; Hepes buffer, 5 (pH 7.2). Salubrinal was purchased from Santa Cruz. The dose of each drug was selected based on previously published studies (1 1 , 20). Caffeine (Sigma) was freshly prepared in water. Additional reagents included: Lipofectamine 2000, TRIzol, Opti-MEM, priopidium iodide, and protein A beads (Invitrogen), MG-132 (BioMol), Trypan blue solution (0.4%; VWR), and tunicamycin (Sigma).

Cell Culture, Transduction, and Electroporations

PC12 and HEK293 cells were maintained in a humidified 5% CO2 and 95% air atmosphere at 37 °C in Dulbecco's Modified Eagle Medium (DMEM) high glucose medium supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 0.05 mg/ml streptomycin (16, 20). PC12 cell lines expressing the human wild-type and mutant aSyn were generated using a tetracycline (Tet)-on system. For the induction of aSyn expression, culture medium was replaced every other day with DMEM containing 1 % horse and 0.5% fetal bovine sera (InVitrogen), 100 ng/ml nerve growth factor (Upstate) and Tet (2 μΜ; Sigma). In some studies, non-induced clones were transduced with recombinant adeno-associated viral (rAAV) particles prior to induction with Tet. Transient transfection was carried out using the Neon transfection system according to the manufacturer's instructions (Invitrogen). PC12 cells (1 -2 x 107/ ml) were transfected by electroporation with 4-8 g of empty vector, wild-type aSyn, or mutant aSyn (gift from Dr. R.G. Perez, Department of

Neurology, University of Pittsburgh) using the following optimized conditions: 1400 V, 20 ms and 1 pulse. The transfection efficiency following electroporation with wild-type aSyn-GFP was -70%.

Ectopic Expression of Herp The Herp and AUBL-Herp constructs have been inserted into a rAAV expression construct (GenDetect). The resulting cDNAs were cloned into the Hindlll/BamHI site of the pAd-YC2 shuttle vector. For homologous recombination, the shuttle vector (5 g) and rescue vector pJM17 (5 g) were co-transfected into HEK293 cells. To amplify the recombinants, cell culture supernantant was serially diluted into serum-free media and incubated with HEK293 cells. The recombinants were purified from supernatants by ultracentrifugation. The band containing mature viral particles were collected and desalted against phosphate-buffered saline (PBS) in a Vivaspin column (Vivascience AG), and titers were determined by counting the number of plaques. Cells were infected with the virus at a MOI of 500 in medium containing 2% FBS for 4 h, after which DMEM containing 10% FBS was added. Analysis of rAAV-GFP expression indicated an infection rate of -85-90%.

Experimental Treatments

To induce ER stress, cultures of PC12 cells were treated with 20 μg/ml Tuni. In some studies, the proteasomal inhibitor MG-132 (0.1 -10 μΜ) or salubrinal (75 μΜ) were added prior to Tuni. These drugs were prepared in DMSO immediately before applying them to the cultures. When DMSO was used as the solvent, their final concentration did not exceed 0.1 %. At the end of each treatment, the cultures were processed for immunoblotting and evaluating cell viability. RNA interference (RNAi)

Cells were transfected with Mission predesigned siRNA duplexes (Sigma) targeting Herp, IP3R1 , RYR1 , and RYR3, or a control siRNA (siRNA-Con; Ambion) using Lipofectamine 2000 (Invitrogen) in Opti-MEM according to manufacturer's protocol. The target sequences of each siRNA are listed in Supplemental Tables 1 S provided in Belal et al., Hum Mol Genet, 2012, Mar 1 ;21 (5):963-77.. Results of quantitative RT-PCR analysis of total RNA from PC12 cells and tissue samples revealed expression of IP3R2, IP3R3 and RYR2 below the limit of detection of the qRT- PCR assay method (Ct values >35). The optimized siRNA concentrations are 100 nM of siRNA-Herp, 250 nM of siRNA- IP3R1 , and 100 nM of each siRNA-RYR1 and siRNA- RYR3 added in combination. After 4 h of transfection, the medium was replaced, and 24-48 h later, the indicated experiments were conducted. To monitor knockdown, cells were harvested and processed for qRT-PCR and Western blot analyses. The

transfection efficiency of siRNA-Con-FITC (Santa Cruz) in PC12 cells was greater than 95% (data not shown).

Assessment of Cell Death

Cell death was assessed by either trypan blue exclusion or propidium iodide staining as described previously (16, 20). Trypan blue and propidium iodide (50 pg/ml) stain only the cells with disrupted plasma membrane integrity so these cells were considered dead. The PI was excited with the 568-nm yellow line of a confocal microscope (Leica), and the acquisition of PI labeling images was performed at the wavelength higher than 600 nm via a photomultiplier through a band-pass filter centered at 605 nm. Dead cells were counted in four microscopic fields per dish, with a minimum of 100 cells per field and results were expressed as a percentage of the total number of cells. All of the experiments were repeated at least three times without knowledge of treatment history.

Immunoprecipitation

Cells and tissues were solubilized in binding buffer containing 50 mM Tris-HCI (pH 7.4), 150 mM NaCI, 1 mM EDTA, 1 mM DTT, 0.2 mM phenylmethanesulfonyl fluoride, and 1 .0% NP-40 as described previously (21 ). The homogenate was centrifuged at 20,000 ^χ g for 10 min. Solubilized proteins were adjusted to 0.1 % NP-40 and incubated for 12 h at 4 °C with a polyclonal antibody to anti-Herp (BioMol), IP3R1 (Millipore) or pan-RyR (Santa Cruz). After an additional incubation with protein A conjugated beads, the immune complexes were then recovered by low speed centrifugation and washed extensively with the binding buffer containing 0.1 % NP-40. Immunoprecipitated proteins were eluted by boiling in SDS-PAGE sampling buffer and analyzed by immunoblotting. Immunoblotting

Protein lysates were centrifuged at 20,000 g and equal amounts of the proteins were loaded into each well of a SDS-PAGE. After electrophoretic separation and transfer to nitrocellulose membranes (Bio-rad), blots were incubated in blocking solution (5% milk in TBS-T) for 1 h at RT, followed by an overnight incubation with primary the following antibodies diluted in blocking buffer: a-Syn [human specific antibody (Abeam) or cross-reactive with human, rat, and mouse (Santa Cruz)], KDEL (Santa Cruz), actin (Sigma), ERK1 (Cell Signaling), caspase-12 (Abeam), Herp [polyclonal antibody

(Biomol) and monoconal antibody (Santa Cruz)], CHOP (Abeam), IP3R1 (Millipore), pan-RyR (Santa Cruz), S5a (Cell Signaling) and presenilin 1 (Abeam). Membranes were then incubated for 1 h in secondary antibody conjugated to horseradish peroxidase (HRP), and bands were visualized by enhanced chemiluminescence (ECL, Thermo- Scientific). Membranes were stripped and re-probed with either the actin or ERK1 antibody to normalize protein loading. The intensity of the signals obtained was quantified by densitometric scanning using Scion (NIH Image).

Immunostaining

Spinal cords were removed after perfusion with heparinized saline (0.9% NaCI) transcardially followed by 4% buffered paraformaldehyde (PFA) and post-fixed overnight in PFA. Serial sections of the lumbar region were sectioned at 30 μιη with a freezing microtome (Microm HM 505 N) and collected on slides. Cultured cells plated on coverslips were fixed for 20 min with 4% paraformaldehyde in PBS following

experimental treatments. Cells were then incubated for 5 min in a solution of 0.2% Triton X-100 in PBS and for 1 h in blocking solution (0.02% Triton X-100, 5% normal horse or goat serum in PBS). Tissue sections and coverslips were processed for immunofluorescence staining as described (16, 21 ) with the following primary

antibodies: aSyn (Abeam), nitro-aSyn (Abeam), Herp (Santa Cruz); CHOP (Cell Signaling); KDEL (Santa Cruz); pan-RyR (Santa Cruz), IP3R1 (Millipore), and NeuN (Millipore). All antibodies were diluted in blocking solution and used within the concentration ranges recommended by the manufacturer. To test for nonspecific staining by the secondary antibodies, additional sections or coverslips were processed in a similar fashion without the primary antibodies or with adsorbed antibodies. After three washes, sections or coverslips were incubated with fluorescein

isothiocyanate(FITC)-conjugated anti-rabbit and Cy3-conjugated anti-mouse secondary antibodies and then mounted. To stain the nuclei, sections or coverslips were further incubated with the nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI) in PBS containing 1 % RNase and 0.2% Triton X-100 for 10 min, and then mounted in

FluorSave aqueous mounting medium (Calbiochem). Immunofluorescence staining was examined by using a NIKON 80i fluorescent microscope equipped with a x60 oil immersion objective lens. For quantification, digitized images of immunostained sections were obtained with Qimaging Retiga 2000 SVGA FAST 1394 cooled digital camera system mounted on the microscope and then analyzed with IP lab software (BD Biosciences- Bio-imaging). Total area of pixel intensity was measured with the automated measurement tools in IP lab software. The total density was averaged and expressed as normalized, corrected vaues.

Measurement of [Ca²*]!^'.

PC12 cells were plated at a density of 1 x106 cells / 35mm glass bottom MatTek dish (Ashland) the day before the experiment. Cells were loaded with 2 μΜ Fura-2 acetoxymethyl ester in Krebs-Ringer-Hepes (KRH) buffer [129mM NaCI, 5mM

NaHCO3, 4.8mM KCI, 1 .2mM KH2PO4, 1 mM CaCI2, 1 .2mM MgCI2, 10 mM glucose and 10mM Hepes (pH7.4)], for 20 minutes and then washed twice with KRH and incubated for additional 30 minutes at 37 °C. Dishes were placed into a heated chamber mounted on the stage of an inverted fluorescence microscope (Nikon Eclipse TiE with perfect focus and DG-5 Xenon excitation) and perfused with Ca²⁺deficient KRH at a rate of 1 .5 ml/ minute. Baseline was established for 6 minutes before stimulation.

Measurements were continued for 4-5 min after Ca²⁺peak was recorded. Fura-2 dual excitation images were captured through a Nikon S Fluor 20X objective (NA 0.75) with a Photometries QuantEM 16bit EMCCD camera using 340 nm and 380 nm excitation filters and a 470-550nm emission filter. Data were acquired and analyzed using Nikon Elements software. Background fluorescence signals were collected at the same rate for the same wavelengths and were subtracted from the corresponding fluorescence images. The fluorescence intensities of 10-20 cells / dish were expressed as ratio of excitation 340/380 nm and area under the curve (AUC).

RT-PCR and Quantitative Real Time-PCR (qRT-PCR) Total RNA was isolated with TRIzol (Invitrogen). To prevent genomic DNA contamination, the isolated total RNA samples were treated with DNAse. 2 g of total RNA was reverse transcribed with Superscript II reverse transcriptase and an oligo(dT) primer (Invitrogen). RT-PCR products were resolved on agorose gels stained with ethidium bromide. Relative quantification of gene expression was performed by normalizing the fluorescence intensities of each band to those of actin. qRT-PCR was performed as previously described (22). The integrity of the RT-PCR products was confirmed by melting curve analysis. Melting curves for all reaction showed one specific peak. We used 18 S rRNA as an endogenous control to normalize variations in RNA extraction and variability in RT efficiency. mRNA levels were quantified with the comparative Ct method (22). The pairs of primers used for RT-PCR and qRT-PCR are listed in Supplemental Tables 2 and 3, respectively, provided in Belal et al., Hum Mol Genet, 2012, Mar 1 ;21 (5):963-77

Animals

Mice transgenic for human A53Ta-Syn (THY1 -SNCA-A53T; Jackson) have been characterized in a previous study (30). All animal experimental procedures were performed in accordance with the guidelines of the NIH and approved by the

Institutional Animal Care and Use Committee at University of Central Florida.

Statistical analysis

Comparison between two groups was performed using Student's f test, whereas multiple comparisons between more than two groups was analyzed by one-way ANOVA and post hoc tests by least significant difference. Data evaluated for the effects of two variables was analyzed using two-way ANOVA (Prism 4 version 4.03; GraphPad Software, Inc.). Results are presented as means ± SEM. For all analyses, statistical significance is defined as a p value of < 0.05.

Calcium imaging

PC12 cells were plated on 35-mm glass bottom dishes (Matek) and loaded with 4 μΜ Fluo-4 acetoxymethyl ester (Invitrogen) in Lock's buffer at 37 deg. C for 30 min. The cells were then washed twice with and incubated in Lock's buffer for an additional 30 min, and then mounted on the stage of an inverted confocal microscope (Carl Zeiss) equipped with a 40χ objective. To trigger ER Ca²⁺ release, 10 μΜ bradykinin was added directly to the cell solution. Cells were excited using the 488-nm laser line, and images were acquired at 5-s intervals under time-lapse mode.

Immunoprecipitation

Cell lysates and tissue homogenates were incubated with an antibody to Herp (BioMol), c-myc (Sigma), S5a (Cell Signaling), IP3R1 (Millipore), pan-RyR (Santa Cruz) or aSyn (Abeam and Santa Cruz) antibody in binding buffer containing 50 mM Tris-HCI (pH 7.4), 150 mM NaCI, 1 mM EDTA, 1 mM DTT, 0.2 mM phenylmethanesulfonyl fluoride, and 1 .0% NP-40. Antigen-antibody complexes were precipitated with

immobilized protein A, washed three times in immunoprecipitation buffer, and

solubilized by heating in Laemmli buffer containing 2-mercaptoethanol at 100 °C for 4 min. The solubilized proteins were separated by electrophoresis and analyzed by immunoblotting.

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PMCID: 2149893.

While various disclosed embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the subject matter disclosed herein can be made in accordance with this Disclosure without departing from the spirit or scope of this Disclosure. In addition, while a particular feature may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.

Thus, the breadth and scope of the subject matter provided in this Disclosure should not be limited by any of the above explicitly described embodiments. Rather, the scope of this Disclosure should be defined in accordance with the following claims and their equivalents.

The terminology used herein is for the purpose of describing particular

embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising." Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

The teachings of any patents, patent applications, technical or scientific articles or other references are incorporated herein in their entirety to the extent not inconsistent with the teachings herein.

Claims

What is claimed is: 1 . A method of stabilizing Ca homeostasis in neurons of a subject, the method comprising administering a therapeutically effective amount of an elF2a phosphatase inhibitor to the subject.

2. The method of claim 1 , wherein the elF2a inhibitor is salubrinal

3. A method of treating or delaying the onset of a neurodegenerative disease

associated with the deregulation of intracellular Ca²⁺ homeostasis in a subject, the method comprising administering to the subject a therapeutically effective amount of an elF2aphosphatase inhibitor.

4. The method of claim 3, wherein said elF2a phosphatase inhibitor is salubrinal.

5. The method of claim 3, wherein said neurodegenerative disease is Alexander disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe disease, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoff disease, Schilder's disease, Sub-Acute Combined Degeneration of the Cord Secondary to Pernicious Anaemia, Schizophrenia, Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease), Spinocerebellar ataxia (multiple types with varying

characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis or Charcot-Marie-Tooth disease.

6. The method of 3, wherein said subject exhibits one or more of the following symptoms:

resting tremor, bradykinesia, muscle rigidity, postural instability, freezing of gait, micrographia, "mask-face", or uncontrolled accelerative movements.

7. The method of claim 3, wherein said subject exhibits one or more of the following symptoms: include memory impairment, disorientation, misinterpreting spatial relationships, impaired speech, personality changes; or impairment of familiar tasks.

8. The method of claim 3, wherein said subject exhibits one or more of the following symptoms: twitching and cramping of the muscles, muscle weakness in the arms or legs, loss of motor control in the arms or legs, general weakness and fatigue, tripping and falling, dropping things, impaired speech, or difficulty chewing or swallowing.

9. The method of claim 3, wherein said neurodegenerative disease is Parkinson's disease, Alzheimer's disease, Huntington's disease, or Amyotrophic lateral sclerosis.

10. A method of treating or delaying the onset of a neurodegenerative disease in a subject in need thereof, said method comprising ameliorating an aberrant accumulation of ER-resident Ca2+ release channels in affected neurons of said subject.

1 1 . The method of claiml O, wherein said ameliorating approach comprises

administering a series of therapeutically effective doses of an elF2a phosphatase inhibitor.

12. The method of claim 10, wherein said elF2a phosphatase inhibitor is salubrinal.

13. The method of 10, wherein said subject exhibits one or more of the following symptoms:

resting tremor, bradykinesia, muscle rigidity, postural instability, freezing of gait, micrographia, "mask-face", or uncontrolled accelerative movements.

14. The method of claim 10, wherein said subject exhibits one or more of the following symptoms: include memory impairment, disorientation, misinterpreting spatial relationships, impaired speech, personality changes; or impairment of familiar tasks.

15. The method of claim 10, wherein said subject exhibits one or more of the following symptoms: twitching and cramping of the muscles, muscle weakness in the arms or legs, loss of motor control in the arms or legs, general weakness and fatigue, tripping and falling, dropping things, impaired speech, or difficulty chewing or swallowing.

16. The method of claim 1 , wherein the inhibitor is provided in a composition further comprising a pharmaceutically acceptable carrier.

17. A composition comprising an elF2a phosphatase inhibitor and a conjunctive agent.

18. The composition of claim 17, further comprising a pharmaceutically acceptable carrier.

19. The composition of claim 17, wherein said conjunctive agent treats or delays onset of Parkinson's disease.

20. The composition of claim 17, wherein said conjunctive agent treats or delays onset of Alzheimer's disease.