Harmful and potentially harmful constituents (HPHCs) in two novel nicotine pouch products in comparison with regular smokeless tobacco products and pharmaceutical nicotine replacement therapy products (NRTs).

BACKGROUND: Tobacco-free nicotine pouches is a novel category of oral nicotine-delivery products. Among current tobacco users such pouches may serve as a low-risk alternative to cigarettes or conventional, tobacco-based oral products e.g., snus and moist snuff. In the United States (U.S.), the market leading nicotine-pouch brand is ZYN®. However, no data on the chemical characteristics of ZYN have been published. METHODS: We screened for 43 compounds potentially present in tobacco products in seven oral nicotine-delivery products: ZYN (dry and moist), snus (General®), moist snuff (CRP2.1 and Grizzly Pouches Wintergreen), and two pharmaceutical, nicotine replacement therapy products (NRTs, Nicorette® lozenge and Nicotinell® gum). Thirty-six of the tested compounds are classified as harmful and potentially harmful constituents (HPHCs) by the Center for Tobacco Products at the U.S. Food and Drug Administration (FDA-CTP). Five additional compounds were included to cover the GOTHIATEK® product standard for Swedish snus and the last two compounds were chosen to include the four primary tobacco specific nitrosamines (TSNAs). RESULTS: The tested products contained nicotine at varying levels. The two ZYN products contained no nitrosamines or polycyclic aromatic hydrocarbons (PAHs) but low levels of ammonia, chromium, formaldehyde, and nickel. In the NRT products we quantified low levels of acetaldehyde, ammonia, cadmium, chromium, lead, nickel, uranium-235, and uranium-238. The largest number (27) and generally the highest levels of HPHCs were quantified in the moist snuff products. For example, they contained six out of seven tested PAHs, and seven out of ten nitrosamines (including NNN and NNK). A total of 19 compounds, none of which were PAHs, were quantified at low levels in the snus product. NNN and NNK levels were five to 12-fold lower in snus compared to the moist snuff products. CONCLUSIONS: No nitrosamines or PAHs were quantified in the ZYN and NRT products. Overall, the number of quantified HPHCs were similar between ZYN and NRT products and found at low levels.

A target-agnostic screen identifies approved drugs to stabilize the endoplasmic reticulum-resident proteome.

Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.

Neuronal Activation Stimulates Cytomegalovirus Promoter-Driven Transgene Expression.

The cytomegalovirus (CMV) immediate early promoter has been extensively developed and exploited for transgene expression in vitro and in vivo, including human clinical trials. The CMV promoter has long been considered a stable, constitutive, and ubiquitous promoter for transgene expression. Using two different CMV-based promoters, we found an increase in CMV-driven transgene expression in the rodent brain and in primary neuronal cultures in response to methamphetamine, glutamate, kainic acid, and activation of G protein-coupled receptor signaling using designer receptors exclusively activated by designer drugs (DREADDs). In contrast, promoters derived from human synapsin 1 (hSYN1) gene or elongation factor 1α (EF1α) did not exhibit altered transgene expression in response to the same neuronal stimulations. Overall, our results suggest that the long-standing assertion that the CMV promoter confers constitutive expression in neurons should be reevaluated, and future studies should empirically determine the activity of the CMV promoter in a given application.

Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

KDEL Receptors Are Differentially Regulated to Maintain the ER Proteome under Calcium Deficiency.

Retention of critical endoplasmic reticulum (ER) luminal proteins needed to carry out diverse functions (e.g., protein synthesis and folding, lipid metabolism) is mediated through a carboxy-terminal ER retention sequence (ERS) and its interaction with KDEL receptors. Here, we demonstrate that depleting ER calcium causes mass departure of ERS-containing proteins from cells by overwhelming KDEL receptors. In addition, we provide evidence that KDELR2 and KDELR3, but not KDELR1, are unfolded protein response (UPR) genes upregulated as an adaptive response to counteract the loss of ERS-containing proteins, suggesting previously unknown isoform-specific functions of the KDEL receptors. Overall, our findings establish that decreases in ER calcium change the composition of the ER luminal proteome and secretome, which can impact cellular functions and cell viability. The redistribution of the ER proteome from inside the cell to the outside has implications for dissecting the complex relationship of ER homeostasis with diverse disease pathologies.

Extracellular esterase activity as an indicator of endoplasmic reticulum calcium depletion.

CONTEXT: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases. OBJECTIVE: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein. MATERIALS AND METHODS: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity. RESULTS: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity. DISCUSSION: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases. CONCLUSION: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.

Pre-α-pro-GDNF and Pre-ß-pro-GDNF Isoforms Are Neuroprotective in the 6-hydroxydopamine Rat Model of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) is one of the most studied neurotrophic factors. GDNF has two splice isoforms, full-length pre-α-pro-GDNF (α-GDNF) and pre-ß-pro-GDNF (ß-GDNF), which has a 26 amino acid deletion in the pro-region. Thus far, studies have focused solely on the α-GDNF isoform, and nothing is known about the in vivo effects of the shorter ß-GDNF variant. Here we compare for the first time the effects of overexpressed α-GDNF and ß-GDNF in non-lesioned rat striatum and the partial 6-hydroxydopamine lesion model of Parkinson's disease. GDNF isoforms were overexpressed with their native pre-pro-sequences in the striatum using an adeno-associated virus (AAV) vector, and the effects on motor performance and dopaminergic phenotype of the nigrostriatal pathway were assessed. In the non-lesioned striatum, both isoforms increased the density of dopamine transporter-positive fibers at 3 weeks after viral vector delivery. Although both isoforms increased the activity of the animals in cylinder assay, only α-GDNF enhanced the use of contralateral paw. Four weeks later, the striatal tyrosine hydroxylase (TH)-immunoreactivity was decreased in both α-GDNF and ß-GDNF treated animals. In the neuroprotection assay, both GDNF splice isoforms increased the number of TH-immunoreactive cells in the substantia nigra but did not promote behavioral recovery based on amphetamine-induced rotation or cylinder assays. Thus, the shorter GDNF isoform, ß-GDNF, and the full-length α-isoform have comparable neuroprotective efficacy on dopamine neurons of the nigrostriatal circuitry.

Combination of CDNF and Deep Brain Stimulation Decreases Neurological Deficits in Late-stage Model Parkinson's Disease.

Several neurotrophic factors (NTF) are shown to be neuroprotective and neurorestorative in pre-clinical animal models for Parkinson's disease (PD), particularly in models where striatal dopamine neuron innervation partially exists. The results of clinical trials on late-stage patients have been modest. Subthalamic deep brain stimulation (STN DBS) is a proven treatment for a selected group of advanced PD patients. The cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic protein, but its effects in animal models of late-stage PD have remained under-researched. The interactions of NTF and STN DBS treatments have not been studied before. We found that a nigral CDNF protein alone had only a marginal effect on the behavioral deficits in a late-stage hemiparkinsonian rat model (6-OHDA MFB). However, CDNF improved the effect of acute STN DBS on front limb use asymmetry at 2 and 3 weeks after CDNF injection. STN lesion-modeling chronic stimulation-had an additive effect in reducing front limb use in the cylinder test and apomorphine-induced rotation. The combination of CDNF and acute STN DBS had a favorable effect on striatal tyrosine hydroxylase. This study presents a novel additive beneficial effect of NTF and STN DBS, which might be explained by the interaction of DBS-induced endogenous NTFs and exogenously injected CDNF. SNpc can be reached via similar trajectories used in clinical STN DBS, and this interaction is an important area for future studies.

Surge of Peripheral Arginine Vasopressin in a Rat Model of Birth Asphyxia.

Mammalian birth is accompanied by a period of obligatory asphyxia, which consists of hypoxia (drop in blood O2 levels) and hypercapnia (elevation of blood CO2 levels). Prolonged, complicated birth can extend the asphyxic period, leading to a pathophysiological situation, and in humans, to the diagnosis of clinical birth asphyxia, the main cause of hypoxic-ischemic encephalopathy (HIE). The neuroendocrine component of birth asphyxia, in particular the increase in circulating levels of arginine vasopressin (AVP), has been extensively studied in humans. Here we show for the first time that normal rat birth is also accompanied by an AVP surge, and that the fetal AVP surge is further enhanced in a model of birth asphyxia, based on exposing 6-day old rat pups to a gas mixture containing 4% O2 and 20% CO2 for 45 min. Instead of AVP, which is highly unstable with a short plasma half-life, we measured the levels of copeptin, the C-terminal part of prepro-AVP that is biochemically much more stable. In our animal model, the bulk of AVP/copeptin release occurred at the beginning of asphyxia (mean 7.8 nM after 15 min of asphyxia), but some release was still ongoing even 90 min after the end of the 45 min experimental asphyxia (mean 1.2 nM). Notably, the highest copeptin levels were measured after hypoxia alone (mean 14.1 nM at 45 min), whereas copeptin levels were low during hypercapnia alone (mean 2.7 nM at 45 min), indicating that the hypoxia component of asphyxia is responsible for the increase in AVP/copeptin release. Alternating the O2 level between 5 and 9% (CO2 at 20%) with 5 min intervals to mimic intermittent asphyxia during prolonged labor resulted in a slower but quantitatively similar rise in copeptin (peak of 8.3 nM at 30 min). Finally, we demonstrate that our rat model satisfies the standard acid-base criteria for birth asphyxia diagnosis, namely a drop in blood pH below 7.0 and the formation of a negative base excess exceeding -11.2 mmol/l. The mechanistic insights from our work validate the use of the present rodent model in preclinical work on birth asphyxia.

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Elevates Stimulus-Evoked Release of Dopamine in Freely-Moving Rats.

Neurotrophic factors (NTFs) hold potential as disease-modifying therapies for neurodegenerative disorders like Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF), and mesencephalic astrocyte-derived neurotrophic factor (MANF) have shown neuroprotective and restorative effects on nigral dopaminergic neurons in various animal models of Parkinson's disease. To date, however, their effects on brain neurochemistry have not been compared using in vivo microdialysis. We measured extracellular concentration of dopamine and activity of dopamine neurochemistry-regulating enzymes in the nigrostriatal system of rat brain. NTFs were unilaterally injected into the striatum of intact Wistar rats. Brain microdialysis experiments were performed 1 and 3 weeks later in freely-moving animals. One week after the treatment, we observed enhanced stimulus-evoked release of dopamine in the striatum of MANF-treated rats, but not in rats treated with GDNF or CDNF. MANF also increased dopamine turnover. Although GDNF did not affect the extracellular level of dopamine, we found significantly elevated tyrosine hydroxylase (TH) and catechol-O-methyltransferase (COMT) activity and decreased monoamine oxidase A (MAO-A) activity in striatal tissue samples 1 week after GDNF injection. The results show that GDNF, CDNF, and MANF have divergent effects on dopaminergic neurotransmission, as well as on dopamine synthetizing and metabolizing enzymes. Although the cellular mechanisms remain to be clarified, knowing the biological effects of exogenously administrated NTFs in intact brain is an important step towards developing novel neurotrophic treatments for degenerative brain diseases.

High fat diet disrupts endoplasmic reticulum calcium homeostasis in the rat liver.

BACKGROUND & AIMS: Disruption to endoplasmic reticulum (ER) calcium homeostasis has been implicated in obesity, however, the ability to longitudinally monitor ER calcium fluctuations has been challenging with prior methodologies. We recently described the development of a Gaussia luciferase (GLuc)-based reporter protein responsive to ER calcium depletion (GLuc-SERCaMP) and investigated the effect of a high fat diet on ER calcium homeostasis. METHODS: A GLuc-based reporter cell line was treated with palmitate, a free fatty acid. Rats intrahepatically injected with GLuc-SERCaMP reporter were fed a cafeteria diet or high fat diet. The liver and plasma were examined for established markers of steatosis and compared to plasma levels of SERCaMP activity. RESULTS: Palmitate induced GLuc-SERCaMP release in vitro, indicating ER calcium depletion. Consumption of a cafeteria diet or high fat pellets correlated with alterations to hepatic ER calcium homeostasis in rats, shown by increased GLuc-SERCaMP release. Access to ad lib high fat pellets also led to a corresponding decrease in microsomal calcium ATPase activity and an increase in markers of hepatic steatosis. In addition to GLuc-SERCaMP, we have also identified endogenous proteins (endogenous SERCaMPs) with a similar response to ER calcium depletion. We demonstrated the release of an endogenous SERCaMP, thought to be a liver esterase, during access to a high fat diet. Attenuation of both GLuc-SERCaMP and endogenous SERCaMP was observed during dantrolene administration. CONCLUSIONS: Here we describe the use of a reporter for in vitro and in vivo models of high fat diet. Our results support the theory that dietary fat intake correlates with a decrease in ER calcium levels in the liver and suggest a high fat diet alters the ER proteome. Lay summary: ER calcium dysregulation was observed in rats fed a cafeteria diet or high fat pellets, with fluctuations in sensor release correlating with fat intake. Attenuation of sensor release, as well as food intake was observed during administration of dantrolene, a drug that stabilizes ER calcium. The study describes a novel technique for liver research and provides insight into cellular processes that may contribute to the pathogenesis of obesity and fatty liver disease.

Longitudinal monitoring of Gaussia and Nano luciferase activities to concurrently assess ER calcium homeostasis and ER stress in vivo.

The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism and calcium storage. The ability to longitudinally monitor ER homeostasis in the same organism would offer insight into progressive molecular and cellular adaptations to physiologic or pathologic states, but has been challenging. We recently described the creation of a Gaussia luciferase (GLuc)-based secreted ER calcium-modulated protein (SERCaMP or GLuc-SERCaMP) to longitudinally monitor ER calcium homeostasis. Here we describe a complementary tool to measure the unfolded protein response (UPR), utilizing a UPRE-driven secreted Nano luciferase (UPRE-secNLuc) to examine the activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1) pathways of the UPR. We observed an upregulation of endogenous ATF6- and XBP1-regulated genes following pharmacologically-induced ER stress that was consistent with responsiveness of the UPRE sensor. Both GLuc and NLuc-based reporters have favorable properties for in vivo studies, however, they are not easily used in combination due to overlapping substrate activities. We describe a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. This study is the first to describe the in vivo measurement of UPRE activation by sampling blood, using an approach that allows concurrent interrogation of two components of ER homeostasis.

Evidence for an Additive Neurorestorative Effect of Simultaneously Administered CDNF and GDNF in Hemiparkinsonian Rats: Implications for Different Mechanism of Action.

Parkinson's disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic (DAergic) neurons of the substantia nigra (SN) and the accumulation of intracellular inclusions containing α-synuclein. Current therapies do not stop the progression of the disease, and the efficacy of these treatments wanes over time. Neurotrophic factors (NTFs) are naturally occurring proteins promoting the survival and differentiation of neurons and the maintenance of neuronal contacts. CDNF (cerebral dopamine NTF) and GDNF (glial cell line-derived NTF) are able to protect DAergic neurons against toxin-induced degeneration in experimental models of PD. Here, we report an additive neurorestorative effect of coadministration of CDNF and GDNF in the unilateral 6-hydroxydopamine (6-OHDA) lesion model of PD in rats. NTFs were given into the striatum four weeks after unilateral intrastriatal injection of 6-OHDA (20 µg). Amphetamine-induced (2.5 mg/kg, i.p.) rotational behavior was measured every two weeks. Number of tyrosine hydroxylase (TH)-positive cells from SN pars compacta (SNpc) and density of TH-positive fibers in the striatum were analyzed at 12 weeks after lesion. CDNF and GDNF alone restored the DAergic function, and one specific dose combination had an additive effect: CDNF (2.5µg) and GDNF (1µg) coadministration led to a stronger trophic effect relative to either of the single treatments alone. The additive effect may indicate different mechanism of action for the NTFs. Indeed, both NTFs activated the survival promoting PI3 kinase (PI3K)-Akt signaling pathway, but only CDNF decreased the expression level of tested endoplasmatic reticulum (ER) stress markers ATF6, glucose-regulated protein 78 (GRP78), and phosphorylation of eukaryotic initiation factor 2α subunit (eIF2α).

Update of neurotrophic factors in neurobiology of addiction and future directions.

Drug addiction is a chronic brain disease and drugs of abuse cause long lasting neuroadaptations. Addiction is characterized by the loss of control over drug use despite harmful consequences, and high rates of relapse even after long periods of abstinence. Neurotrophic factors (NTFs) are well known for their actions on neuronal survival in the peripheral nervous system. Moreover, NTFs have been shown to be involved in synaptic plasticity in the brain. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are two of the most studied NTFs and both of them have been reported to increase craving when administered into the mesocorticolimbic dopaminergic system after drug self-administration. Here we review recent data on BDNF and GDNF functions in addiction-related behavior and discuss them in relation to previous findings. Finally, we give an insight into how new technologies could aid in further elucidating the role of these factors in drug addiction.

Synthesis and biological evaluation of novel (123)I-labeled 4-(4-iodophenyl)butanoyl-L-prolyl-(2S)-pyrrolidines for imaging prolyl oligopeptidase in vivo.

Prolyl oligopeptidase (POP) may be associated with neuromodulation and development of neurodegenerative diseases and it was recently shown to participate in the inflammatory cascade along with matrix metalloproteinases. Radiotracers, which can be used for non-invasive imaging, are needed for investigating the role of POP in normal physiology and in pathophysiological conditions in vivo. We synthesized two novel POP-specific (123)I-radiolabeled 4-phenylbutanoyl-L-prolyl-pyrrolidines of which 4-(4-[(123)I]iodophenyl)butanoyl-L-prolyl-2(S)-cyanopyrrolidine ([(123)I]2f, Ki = 4.2 nM) was selected. The selected compound has an electrophilic cyano group that is known to increase the dissociation time of POP inhibitors. [(123)I]2f was synthesized in high radiochemical yield and purity (87 ± 4%, >99%, respectively) and with a specific activity of 456 ± 98 GBq/µmol. [(123)I]2f was evaluated in healthy mice (C57Bl/6JRccHsd) by ex vivo biodistribution studies and SPECT imaging. Pretreatment with the known inhibitor 4-phenylbutanoyl-L-prolyl-(2S)-cyanopyrrolidine (KYP-2047, 2d, Ki = 0.023 nM) showed that binding of [(123)I]2f was POP specific. In addition, [(123)I]2f was evaluated in models of neuroinflammation and acute localized inflammation. A minor increase in binding of [(123)I]2f was observed in the inflamed region in the acute localized inflammation model. Similar increase in binding was not observed in the neuroinflammation model.

High correlation between in vivo [123I]ß-CIT SPECT/CT imaging and post-mortem immunohistochemical findings in the evaluation of lesions induced by 6-OHDA in rats.

BACKGROUND: 6-Hydroxydopamine (6-OHDA) is widely used in pre-clinical animal studies to induce degeneration of midbrain dopamine neurons to create animal models of Parkinson's disease. The aim of our study was to evaluate the potential of combined single-photon emission computed tomography/computed tomography (SPECT/CT) for the detection of differences in 6-OHDA-induced partial lesions in a dose- and time-dependent manner using the dopamine transporter (DAT) ligand 2ß-carbomethoxy-3ß-(4-[123I]iodophenyl)tropane ([123I]ß-CIT). METHODS: Rats were unilaterally lesioned with intrastriatal injections of 8 or 2 × 10 µg 6-OHDA. At 2 or 4 weeks post-lesion, 40 to 50 MBq [123I]ß-CIT was administered intravenously and rats were imaged with small-animal SPECT/CT under isoflurane anesthesia. The striatum was delineated and mean striatal activity in the lesioned side was compared to the intact side. After the [123I]ß-CIT SPECT/CT scan, the rats were tested for amphetamine-induced rotation asymmetry, and their brains were immunohistochemically stained for DAT and tyrosine hydroxylase (TH). The fiber density of DAT- and TH-stained striata was estimated, and TH-immunoreactive cells in the rat substantia nigra pars compacta (SNpc) were stereologically counted. RESULTS: The striatal uptake of [123I]ß-CIT differed significantly between the lesion groups and the results were highly correlated to both striatal DAT- and TH-immunoreactive fiber densities and to TH-immunoreactive cell numbers in the rat SNpc. No clear progression of the lesion could be seen. CONCLUSIONS: [123I]ß-CIT SPECT/CT is a valuable tool in predicting the condition of the rat midbrain dopaminergic pathway in the unilateral partial 6-OHDA lesion model of Parkinson's disease and it offers many advantages, allowing repeated non-invasive analysis of living animals.

Gene therapy with AAV2-CDNF provides functional benefits in a rat model of Parkinson's disease.

Cerebral dopamine neurotrophic factor (CDNF) protein has been shown to protect the nigrostriatal dopaminergic pathway when given as intrastriatal infusions in rat and mouse models of Parkinson's disease (PD). In this study, we assessed the neuroprotective effect of CDNF delivered with a recombinant adeno-associated viral (AAV) serotype 2 vector in a rat 6-hydroxydopamine (6-OHDA) model of PD. AAV2 vectors encoding CDNF, glial cell line-derived neurotrophic factor (GDNF), or green fluorescent protein were injected into the rat striatum. Protein expression analysis showed that our AAV2 vector efficiently delivered the neurotrophic factor genes into the brain and gave rise to a long-lasting expression of the proteins. Two weeks after AAV2 vector injection, 6-OHDA was injected into the rat striatum, creating a progressive degeneration of the nigrostriatal dopaminergic system. Treatment with AAV2-CDNF resulted in a marked decrease in amphetamine-induced ipsilateral rotations while it provided only partial protection of tyrosine hydroxylase (TH)-immunoreactive cells in the rat substantia nigra pars compacta and TH-reactive fibers in the striatum. Results from this study provide additional evidence that CDNF can be considered a potential treatment of Parkinson's disease.

Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease.

Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) constitute a novel, evolutionarily conserved family of neurotrophic factors (NTF) expressed in vertebrates and invertebrates. The effects of two-week infusions of CDNF, MANF and glial cell line-derived neurotrophic factor (GDNF) were studied in a rat 6-hydroxydopamine (6-OHDA) hemiparkinsonian model. Degeneration of nigrostriatal dopamine nerve tract after toxin injection was assessed by measuring amphetamine-induced rotational behavior, and at the end of the experiment by quantifying tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc) and TH-positive fibers in the striatum. The diffusion of the NTFs into the brain tissue following chronic infusion was also studied. Finally, we examined the transportation of intrastriatally injected (125)I-CDNF within the brain. The amphetamine-induced rotational behavior was gradually normalized in rats treated with CDNF for two weeks following the intrastriatal 6-OHDA injection. CDNF was also able to inhibit 6-OHDA-induced loss of TH-immunoreactive cells of the SNpc and TH-positive fibers in the striatum. MANF and GDNF had no statistically significant effect in any of the above measures. The volume of distribution for MANF in the striatum was significantly larger than that of GDNF after 3-day infusions. Both (125)I-CDNF and (125)I-GDNF were retrogradely transported from the striatum to the SN. No behavioral signs of toxicity were observed during treatment with the three NTFs. These results imply that CDNF may have potential as a neuroprotective or even neurorestorative therapy of PD.

Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson's disease.

Neurotrophic factors are promising candidates for the treatment of Parkinson's disease (PD). Mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to a novel evolutionarily conserved family of neurotrophic factors. We examined whether MANF has neuroprotective and neurorestorative effect in an experimental model of PD in rats. We also studied the distribution and transportation of intrastriatally injected MANF in the brain and compared it with glial cell line-derived neurotrophic factor (GDNF). Unilateral lesion of nigrostriatal dopaminergic system was induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA). Amphetamine-induced turning behavior was monitored up to 12 weeks after the unilateral lesion. The local diffusion at the injection site and transportation profiles of intrastriatally injected MANF and GDNF were studied by immunohistochemical detection of the unlabeled growth factors as well as by autoradiographic and gamma counting detection of (125)I-labeled trophic factors. Intrastriatally injected MANF protected nigrostriatal dopaminergic nerves from 6-OHDA-induced degeneration as evaluated by counting tyrosine hydroxylase (TH)-positive cell bodies in the substantia nigra (SN) and TH-positive fibers in the striatum. More importantly, MANF also restored the function of the nigrostriatal dopaminergic system when administered either 6 h before or 4 weeks after 6-OHDA administration in the striatum. MANF was distributed throughout the striatum more readily than GDNF. The mechanism of MANF action differs from that of GDNF because intrastriatally injected (125)I-MANF was transported to the frontal cortex, whereas (125)I-GDNF was transported to the SN. Our results suggest that MANF is readily distributed throughout the striatum and has significant therapeutic potential for the treatment of PD.