Retinoid X Receptor Activation Prevents Diabetic Retinopathy in Murine Models.

Previously, the RXR agonist UAB126 demonstrated therapeutic potential to treat obese mice by controlling blood glucose levels (BGL) and altering the expression of genes associated with lipid metabolism and inflammatory response. The purpose of the study was to assess the effects of UAB126 on the progression of diabetic retinopathy (DR) in rodent models of type 1 diabetes (T1D), streptozotocin-induced, and type 2 diabetes (T2D), in db/db mice. UAB126 treatment was delivered either by oral gavage for 6 weeks or by topical application of eye drops for 2 weeks. At the end of the treatment, the retinal function of diabetic mice was assessed by electroretinography (ERG), and their retinal tissue was harvested for protein and gene expression analyses. Bone-marrow cells were isolated and differentiated into bone marrow-derived macrophages (BMDMs). The glycolysis stress test and the 2-DG glucose uptake analysis were performed. Our results demonstrated that in the UAB126-treated diabetic BMDMs, the ECAR rate and the 2-DG uptake were improved as compared to untreated diabetic BMDMs. In UAB126-treated diabetic mice, hyperglycemia was reduced and associated with the preservation of ERG amplitudes and enhanced AMPK activity. Retinas from diabetic mice treated with topical UAB126 demonstrated an increase in Rxr and Ppar and the expression of genes associated with lipid metabolism. Altogether, our data indicate that RXR activation is beneficial to preclinical models of DR.

GADD34 Ablation Exacerbates Retinal Degeneration in P23H RHO Mice.

The UPR is sustainably activated in degenerating retinas, leading to translational inhibition via p-eIF2α. Recent findings have demonstrated that ablation of growth arrest and DNA damage-inducible protein 34 (GADD34), a protein phosphatase 1 regulatory subunit permitting translational machinery operation through p-eIF2α elevation, does not impact the rate of translation in fast-degenerating rd16 mice. The current study aimed to validate whether P23H RHO mice degenerating at a slower pace manifest translational attenuation and whether GADD34 ablation impacts the rate of retinal degeneration via further suppression of retinal protein synthesis and apoptotic cell death. For this study, mice were examined with ERG and histological analyses. The molecular assessment was conducted in the naïve and LPS-challenged mice using Western blot and qRT-PCR analyses. Thus, this study demonstrates that the P23H RHO retinas manifest translational attenuation. However, GADD34 ablation resulted in a more prominent p-eIF2a increase without impacting the translation rate. GADD34 deficiency also led to a reduction in scotopic ERG amplitudes and an increased number of TUNEL-positive cells. Molecular analysis revealed that GADD34 deficiency reduces the expression of p-STAT3 and Il-6 while increasing the expression of Tnfa. Overall, the data indicate that GADD34 plays a multifunctional role. Under chronic UPR activation, GADD34 acts as a feedback player, dephosphorylating p-eIF2a, although this role does not seem to be critical. Additionally, GADD34 controls cytokine expression and STAT3 activation. Perhaps these molecular events are particularly important in controlling the pace of retinal degeneration.

Tribbles homolog 3-mediated targeting the AKT/mTOR axis in mice with retinal degeneration.

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3-/- retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.

A Novel Tree Shrew Model of Diabetic Retinopathy.

Existing animal models with rod-dominant retinas have shown that hyperglycemia injures neurons, but it is not yet clearly understood how blue cone photoreceptors and retinal ganglion cells (RGCs) deteriorate in patients because of compromised insulin tolerance. In contrast, northern tree shrews (Tupaia Belangeri), one of the closest living relatives of primates, have a cone-dominant retina with short wave sensitivity (SWS) and long wave sensitivity (LWS) cones. Therefore, we injected animals with a single streptozotocin dose (175 mg/kg i.p.) to investigate whether sustained hyperglycemia models the features of human diabetic retinopathy (DR). We used the photopic electroretinogram (ERG) to measure the amplitudes of A and B waves and the photopic negative responses (PhNR) to evaluate cone and RGC function. Retinal flat mounts were prepared for immunohistochemical analysis to count the numbers of neurons with antibodies against cone opsins and RGC specific BRN3a proteins. The levels of the proteins TRIB3, ISR-1, and p-AKT/p-mTOR were measured with western blot. The results demonstrated that tree shrews manifested sustained hyperglycemia leading to a slight but significant loss of SWS cones (12%) and RGCs (20%) 16 weeks after streptozotocin injection. The loss of BRN3a-positive RGCs was also reflected by a 30% decline in BRN3a protein expression. These were accompanied by reduced ERG amplitudes and PhNRs. Importantly, the diabetic retinas demonstrated increased expression of TRIB3 and level of p-AKT/p-mTOR axis but reduced level of IRS-1 protein. Therefore, a new non-primate model of DR with SWS cone and RGC dysfunction lays the foundation to better understand retinal pathophysiology at the molecular level and opens an avenue for improving the research on the treatment of human eye diseases.

Endoplasmic reticulum stress: New insights into the pathogenesis and treatment of retinal degenerative diseases.

Physiological equilibrium in the retina depends on coordinated work between rod and cone photoreceptors and can be compromised by the expression of mutant proteins leading to inherited retinal degeneration (IRD). IRD is a diverse group of retinal dystrophies with multifaceted molecular mechanisms that are not fully understood. In this review, we focus on the contribution of chronically activated unfolded protein response (UPR) to inherited retinal pathogenesis, placing special emphasis on studies employing genetically modified animal models. As constitutively active UPR in degenerating retinas may activate pro-apoptotic programs associated with oxidative stress, pro-inflammatory signaling, dysfunctional autophagy, free cytosolic Ca2+ overload, and altered protein synthesis rate in the retina, we focus on the regulatory mechanisms of translational attenuation and approaches to overcoming translational attenuation in degenerating retinas. We also discuss current research on the role of the UPR mediator PERK and its downstream targets in degenerating retinas and highlight the therapeutic benefits of reprogramming PERK signaling in preclinical animal models of IRD. Finally, we describe pharmacological approaches targeting UPR in ocular diseases and consider their potential applications to IRD.

Role of Translational Attenuation in Inherited Retinal Degeneration.

Purpose: We reported previously that retinas of mice with inherited retinal degeneration make less protein than retinas of normal mice. Despite recent studies suggesting that diminished protein synthesis rates may contribute to neurologic disorders, a direct link between protein synthesis rates and the progression of neurodegeneration has not been established. Moreover, it remains unclear whether reduced protein synthesis could be involved in retinal pathogenesis. Dysregulation of AKT/mTOR signaling has been reported in the retina during retinal degeneration, but to what extent this signaling contributes to translational attenuation in these mice remains uncertain. Methods: C57BL/6J and rd16 mice were subcutaneously injected with anisomycin to chronically inhibit protein synthesis rates. An AAV2 construct encoding constitutively active 4ebp1 was subretinally delivered in wildtype animals to lower protein synthesis rates. 4ebp1/2 were knocked out in rd16 mice. Results: Anisomycin treatment lowered retinal translation rates, accelerated retinal degeneration in rd16 mice, and initiated cell death in the retinas of C57BL/6J mice. AAV-mediated transfer of constitutively active 4ebp1-4A into the subretinal space of wildtype animals inhibited protein synthesis, and led to reduced electroretinography amplitudes and fewer ONL nuclei. Finally, we report that restoring protein synthesis rates by knocking out 4ebp1/2 was associated with an approximately 2-fold increase in rhodopsin levels and a delay in retinal degeneration in rd16 mice. Conclusions: Our study indicates that protein synthesis inhibition is likely not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but may be harmful to degenerating retinas, and that restoring protein synthesis may have therapeutic potential in delaying the progression of retinal degeneration.

Biodistribution of adeno-associated virus type 2 with mutations in the capsid that contribute to heparan sulfate proteoglycan binding.

We compared the phenotypes of three mutant AAV2 viruses containing mutations in arginine amino acids (R585, R588 and R484) previously shown to be involved in AAV2 heparan sulfate binding. The transduction efficiencies of wild type and mutant viruses were determined in the eye, the brain and peripheral organs following subretinal, striatal and intravenous injection, respectively, in mice and rats. We found that each of the three mutants (the single mutant R585A; the double mutant R585, 588A; and the triple mutant R585, 588, 484A) had a unique phenotype compared to wt and each other. R585A was completely defective for transducing peripheral organs via intravenous injection, suggesting that R585A may be useful for targeting peripheral organs by substitution of peptide ligands in the capsid surface. In the brain, all three mutants displayed widespread transduction, with the double mutant R585, 588A displaying the greatest spread and the greatest number of transduced neurons. The double mutant was also extremely efficient for retrograde transport, while the triple mutant was almost completely defective for retrograde transport. This suggested that R484 may be directly involved in interaction with the transport machinery. Finally, the double mutant also displayed improved transduction of the eye compared to wild type and the other mutants.

The Lipase Activity of Phospholipase D2 is Responsible for Nigral Neurodegeneration in a Rat Model of Parkinson's Disease.

Phospholipase D2 (PLD2), an enzyme involved in vesicle trafficking and membrane signaling, interacts with α-synuclein, a protein known to contribute in the development of Parkinson disease (PD). We previously reported that PLD2 overexpression in rat substantia nigra pars compacta (SNc) causes a rapid neurodegeneration of dopamine neurons, and that α-synuclein suppresses PLD2-induced nigral degeneration (Gorbatyuk et al., 2010). Here, we report that PLD2 toxicity is due to its lipase activity. Overexpression of a catalytically inactive mutant (K758R) of PLD2 prevents the loss of dopaminergic neurons in the SNc and does not show signs of toxicity after 10 weeks of overexpression. Further, mutant K758R does not affect dopamine levels in the striatum. In contrast, mutants that prevent PLD2 interaction with dynamin or growth factor receptor bound protein 2 (Grb2) but retained lipase activity, continued to show rapid neurodegeneration. These findings suggest that neither the interaction of PLD2 with dynamin, which has a role in vesicle trafficking, nor the PLD2 interaction with Grb2, which has multiple roles in cell cycle control, chemotaxis and activation of tyrosine kinase complexes, are the primary cause of neurodegeneration. Instead, the synthesis of phosphatidic acid (the product of PLD2), which is a second messenger in multiple cellular pathways, appears to be the key to PLD2 induced neurodegeneration. The fact that α-synuclein is a regulator of PLD2 activity suggests that regulation of PLD2 activity could be important in the progression of PD.

Up-regulation of activating transcription factor 4 induces severe loss of dopamine nigral neurons in a rat model of Parkinson's disease.

Activating transcription factor 4 (ATF4) is a member of the PERK signaling pathway, which directly binds endoplasmic reticulum stress target genes and plays a crucial role in both adaptations to stress and activation of apoptosis. Previous publications demonstrated conflicting evidence on the role of ATF4 in the pathogenesis of neurodegenerative disorders. In this study, we used recombinant adeno-associate virus (rAAV)-mediated gene transfer to investigate if the sustained up-regulation of ATF4 launches a pro-survival or pro-death trend in the dopamine (DA) cells of the substantia nigra pars compacta in a rat model of Parkinson-like neurodegeneration induced by human alpha-synuclein (αS) overexpression. We showed that ATF4 does not protect nigral DA neurons against an αS-induced pathology. Moreover, the rAAV-mediated overexpression of ATF4 resulted in severe nigra-striatal degeneration via activation of caspases 3/7.

The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons.

Age-related structural changes and gradual loss of key enzymes significantly affect the ability of the endoplasmic reticulum (ER) to facilitate proper protein folding and maintain homeostasis. In this work, we present several lines of evidence supporting the hypothesis that the age-related decline in expression of the ER chaperone glucose-regulated protein 78 (GRP78) could be related to the development of Parkinson's disease. We first determined that old (24 months) rats exhibit significantly lower levels of GRP78 protein in the nigrostriatal system as compared with young (2 months) animals. Then using recombinant adeno-associate virus-mediated gene transfer, we found that GRP78 downregulation by specific small interfering RNAs (siRNAs) aggravates alpha-synuclein (α-syn) neurotoxicity in nigral dopamine (DA) neurons. Moreover, the degree of chaperone decline corresponds with the severity of neurodegeneration. Additionally, comparative analysis of nigral tissues obtained from old and young rats revealed that aging affects the capacity of nigral DA cells to upregulate endogenous GRP78 protein in response to human α-syn neurotoxicity. Finally, we demonstrated that a sustained increase of GRP78 protein over the course of 9 months protected aging nigral DA neurons in the α-syn-induced rat model of Parkinson's-like neurodegeneration. Our data indicate that the ER chaperone GRP78 may have therapeutic potential for preventing and/or slowing age-related neurodegeneration.

Salivary peptide tyrosine-tyrosine 3-36 modulates ingestive behavior without inducing taste aversion.

Hormone peptide tyrosine-tyrosine (PYY) is secreted into circulation from the gut L-endocrine cells in response to food intake, thus inducing satiation during interaction with its preferred receptor, Y2R. Clinical applications of systemically administered PYY for the purpose of reducing body weight were compromised as a result of the common side effect of visceral sickness. We describe here a novel approach of elevating PYY in saliva in mice, which, although reliably inducing strong anorexic responses, does not cause aversive reactions. The augmentation of salivary PYY activated forebrain areas known to mediate feeding, hunger, and satiation while minimally affecting brainstem chemoreceptor zones triggering nausea. By comparing neuronal pathways activated by systemic versus salivary PYY, we identified a metabolic circuit associated with Y2R-positive cells in the oral cavity and extending through brainstem nuclei into hypothalamic satiety centers. The discovery of this alternative circuit that regulates ingestive behavior without inducing taste aversion may open the possibility of a therapeutic application of PYY for the treatment of obesity via direct oral application.

The Molecular Chaperone GRP78/BiP as a Therapeutic Target for Neurodegenerative Disorders: A Mini Review.

The glucose regulated protein 78 (GRP78), also known as BiP, is the endoplasmatic reticulum (ER) homologue of HSP70, which plays a dual role in the ER by controlling protein folding, in order to prevent aggregation, and by regulating the signaling of the unfolded protein response (UPR). Most neurodegenerative disorders including Parkinson's, Alzheimer's diseases and progressive retinal degeneration are characterized by activation of the UPR and modified expression of GRP78. The expression levels and activity of GRP78 are altered with age raising the question of whether the lack of GRP78 could be a predisposing factor for many neurodegenerative disorders associated with age including PD, Alzheimer and Age-related macular degeneration. Attempts to induce or upregulate GRP78 in animal models of neurodegeneration have recently been made with the help of pharmacological BiP protein Inducer X (BIX) and GRP78 cDNA delivery via adeno-associated virus (AAV) vectors. The results of these studies validate GRP78 as a new therapeutic target for treatments of forebrain ischemia, Parkinson disease and retinal degeneration. These data, together with the results from age-related studies, highlight the importance for developing drugs to induce elevation of endogenous GRP78 in order to increase cellular survival and extend functional longevity.

ER stress is involved in T17M rhodopsin-induced retinal degeneration.

PURPOSE: The human rhodopsin (Rho) mutation T17M leads to autosomal dominant retinitis pigmentosa (adRP). The goal of our study was to elucidate the role of endoplasmic reticulum (ER) stress in retinal degeneration in hT17M Rho mice and identify potential candidates for adRP gene therapy. METHODS: We used transgenic mice expressing the ER stress-activated indicator (ERAI) and hT17M Rho to evaluate the activation of ER stress responses. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze changes in the expression of 30 unfolded protein response (UPR)-associated genes at P12, 15, 18, 21, and 25. The cytosolic fraction of hT17M Rho retinal cells was used to measure the release of cytochrome C and apoptotic inducing factor-1 (AIF1) by Western blotting. Optical coherence tomography (OCT) analysis was performed for 1-month-old hT17M Rho mice. RESULTS: hT17M Rho was localized in the outer nuclear layer (ONL) of T17M(+/-)ERAI(+/-) photoreceptors as well as C57BL/6 retinas injected with AAV-hT17M Rho-GFP. In P15 hT17M Rho retinas, we observed an up-regulation of UPR genes (Atf4, Eif2α, Xbp1, Bip, Canx, and Hsp90), autophagy genes and proapoptotic Bcl2 genes. OCT, and the downregulation of Nrl and Crx gene expression confirmed that cell death occurs in 55% of photoreceptors via the up-regulation of caspase-3 and caspase-12, and the release of AIF1 from the mitochondria. CONCLUSIONS: The ER stress response is involved in retinal degeneration in hT17M Rho mice. The final demise of photoreceptors occurs via apoptosis involving ER stress-associated and mitochondria-induced caspase activation. We identified Atg5, Atg7, Bax, Bid, Bik, and Noxa as potential therapeutic targets for adRP treatment.

Glucose regulated protein 78 diminishes α-synuclein neurotoxicity in a rat model of Parkinson disease.

Accumulation of human wild-type (wt) α-synuclein (α-syn) induces neurodegeneration in humans and in experimental rodent models of Parkinson disease (PD). It also leads to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). We overexpressed glucose regulated protein 78, also known as BiP (GRP78/BiP), to test the hypothesis that this ER chaperone modulates the UPR, blocks apoptosis, and promotes the survival of nigral dopamine (DA) neurons in a rat model of PD induced by elevated level of human α-syn. We determined that α-syn activates ER stress mediators associated with pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) signaling pathways as well as proaoptotic CCAAT/-enhancer-binding protein homologous protein (CHOP) in nigral DA neurons. At the same time, overexpression of GRP78/BiP diminished α-syn neurotoxicity by down regulating ER stress mediators and the level of apoptosis, promoted survival of nigral tyrosine hydroxylase (TH) positive cells and resulted in higher levels of striatal DA, while eliminating amphetamine induced behavioral asymmetry. We also detected a complex between GRP78/BiP and α-syn that may contribute to prevention of the neurotoxicity caused by α-syn. Our data suggest that the molecular chaperone GRP78/BiP plays a neuroprotective role in α-syn-induced Parkinson-like neurodegeneration.

α-Synuclein expression in rat substantia nigra suppresses phospholipase D2 toxicity and nigral neurodegeneration.

We present genetic evidence that an in vivo role of α-synuclein (α-syn) is to inhibit phospholipase D2 (PLD2), an enzyme that is believed to participate in vesicle trafficking, membrane signaling, and both endo- and exocytosis. Overexpression of PLD2 in rat substantia nigra pars compacta (SNc) caused severe neurodegeneration of dopamine (DA) neurons, loss of striatal DA, and an associated ipsilateral amphetamine-induced rotational asymmetry. Coexpression of human wild type α-syn suppressed PLD2 neurodegeneration, DA loss, and amphetamine-induced rotational asymmetry. However, an α-syn mutant defective for inhibition of PLD2 in vitro also failed to inhibit PLD toxicity in vivo. Further, reduction of PLD2 activity in SNc, either by siRNA knockdown of PLD2 or overexpression of α-syn, both produced an unusual contralateral amphetamine-induced rotational asymmetry, opposite to that seen with overexpression of PLD2, suggesting that PLD2 and α-syn were both involved in DA release or reuptake. Finally, α-syn coimmunoprecipitated with PLD2 from extracts prepared from striatal tissues. Taken together, our data demonstrate that α-syn is an inhibitor of PLD2 in vivo, and confirm earlier reports that α-syn inhibits PLD2 in vitro. Our data also demonstrate that it is possible to use viral-mediated gene transfer to study gene interactions in vivo.

In vivo RNAi-mediated alpha-synuclein silencing induces nigrostriatal degeneration.

Two small-interfering RNAs (siRNAs) targeting alpha-synuclein (alpha-syn) and three control siRNAs were cloned in an adeno-associated virus (AAV) vector and unilaterally injected into rat substantia nigra pars compacta (SNc). Reduction of alpha-syn resulted in a rapid (4 week) reduction in the number of tyrosine hydroxylase (TH) positive cells and striatal dopamine (DA) on the injected side. The level of neurodegeneration induced by the different siRNAs correlated with their ability to downregulate alpha-syn protein and mRNA in tissue culture and in vivo. Examination of various SNc neuronal markers indicated that neurodegeneration was due to cell loss and not just downregulation of DA synthesis. Reduction of alpha-syn also resulted in a pronounced amphetamine induced behavioral asymmetry consistent with the level of neurodegeneration. In contrast, none of the three control siRNAs, which targeted genes not normally expressed in SNc, showed evidence of neurodegeneration or behavioral asymmetry, even at longer survival times. Moreover, co-expression of both rat alpha-syn and alpha-syn siRNA partially reversed the neurodegenerative and behavioral effects of alpha-syn siRNA alone. Our data show that alpha-syn plays an important role in the rat SNc and suggest that both up- and downregulation of wild-type alpha-syn expression increase the risk of nigrostriatal pathology.

Restoration of visual function in P23H rhodopsin transgenic rats by gene delivery of BiP/Grp78.

The P23H mutation within the rhodopsin gene (RHO) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2alpha and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2alpha, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

Nigrostriatal rAAV-mediated GDNF overexpression induces robust weight loss in a rat model of age-related obesity.

Intraventricular administration of glial cell line-derived neurotrophic factor (GDNF) in primate and humans to study Parkinson's disease (PD) has revealed the potential for GDNF to induce weight loss. Our previous data indicate that bilateral continuous hypothalamic GDNF overexpression via recombinant adeno-associated virus (rAAV) results in significant failure to gain weight in young rats and weight loss in aged rats. Based on these previous results, we hypothesized that because the nigrostriatal tract passes through the lateral hypothalamus, motor hyperactivity mediated by nigrostriatal dopamine (DA) may have been responsible for the previously observed effect on body weight. In this study, we compared bilateral injections of rAAV2/5-GDNF in hypothalamus versus substantia nigra (SN) in aged Brown-Norway X Fisher 344 rats. Nigrostriatal GDNF overexpression resulted in significantly greater weight loss than rats treated in hypothalamus. The nigral or hypothalamic GDNF-induced weight loss was unrelated to motor activity levels of the rats, though some of the weight loss could be attributed to a transient reduction in food intake. Forebrain DA levels did not account for the observed effects on body weight, although GDNF-induced increases in nucleus accumbens DA may have partially contributed to this effect in the hypothalamic GDNF-treated group. However, only nigrostriatal GDNF overexpression induced activation of phosphorylated extracellular signal-regulated kinase (p-ERK) in a small population of corticotrophin-releasing factor [corticotrophin-releasing hormone (CRH)] neurons located specifically in the medial parvocellullar division (MPD) of the paraventricular nucleus of the hypothalamus. Activation of these hypothalamic CRH neurons likely accounted for the observed metabolic effects leading to weight loss in obese rats.

The phosphorylation state of Ser-129 in human alpha-synuclein determines neurodegeneration in a rat model of Parkinson disease.

Studies have shown that alpha-synuclein (alpha-syn) deposited in Lewy bodies in brain tissue from patients with Parkinson disease (PD) is extensively phosphorylated at Ser-129. We used recombinant Adeno-associated virus (rAAV) to overexpress human wild-type (wt) alpha-syn and two human alpha-syn mutants with site-directed replacement of Ser-129 to alanine (S129A) or to aspartate (S129D) in the nigrostriatal tract of the rat to investigate the effect of Ser-129 phosphorylation state on dopaminergic neuron pathology. Rats were injected with rAAV2/5 vectors in the substantia nigra pars compacta (SNc) on one side of the brain; the other side remained as a nontransduced control. The level of human wt or mutant alpha-syn expressed on the injected side was about four times the endogenous rat alpha-syn. There was a significant reduction of dopaminergic neurons in the SNc and dopamine (DA) and tyrosine hydroxylase (TH) levels in the striatum of all S129A-treated rats as early as 4 wk postinjection. Nigral DA pathology occurred more slowly in the wt-injected animals, but by 26 wk the wt alpha-syn group lost nigral TH neurons equivalent to the mutated S129A group at 8 wk. In stark contrast, we did not observe any pathological changes in S129D-treated animals. Therefore, the nonphosphorylated form of S129 exacerbates alpha-syn-induced nigral pathology, whereas Ser-129 phosphorylation eliminates alpha-syn-induced nigrostriatal degeneration. This suggests possible new therapeutic targets for Parkinson Disease.