Rat Tyrosine Hydroxylase Promoter Research Articles

196-OR: (Pro) renin Receptor Signaling in Tyrosine Hydroxylase Neurons in the Hypothalamic Paraventricular Nucleus Is Required for Obesity-Associated Glucose Metabolic Impairment

We recently reported that neuron-specific (pro) renin receptor (PRR) deletion reduces fasting blood glucose (BG) levels and improves glucose tolerance in high fat diet (HFD) -treated mice. To investigate underlying mechanisms, we focused on tyrosine hydroxylase (TH) neurons in the hypothalamic paraventricular nucleus (THPVN neurons) , a largely GABAergic population that expresses PRR and projects to autonomic centers important for BG regulation. To delete PRR from THPVN neurons, we injected into the PVN of PRR-loxp mice either an adeno-associated virus expressing cre recombinase under the control of the rat TH promoter (AAV2-TH-Cre) or AAV2-TH-eGFP as a control. After placing animals on a HFD for 6 wk, we found that the obesity-induced increase of fasting BG levels was blocked by PRR deletion from THPVN neurons (118.0 ± 3.7 vs. 139.3 ± 6.6 mg/dL, n=6/group, p= 0.04) , accompanied by a trend towards improved i.p. glucose tolerance (AUC: 12990 ± 1136 vs. 9467 ± 1298, n=6, p=0.07) . To further investigate the role of THPVN neurons in glucose homeostasis, we employed a Designer Receptors Exclusively Activated by Designer Drugs (DREADD) approach whereby AAV2-TH-Cre was injected bilaterally into the PVN of either hM3Dq-loxp (excitatory) or hM4Di-loxp (inhibitory) mice, and continuous glucose monitoring was performed in conscious, freely moving mice. Following i.p. injection of clozapine-N-oxide (CNO, 1 mg/Kg) or vehicle, we found that whereas basal BG levels were not altered by THPVN neuron activation, THPVN neuron inhibition significantly increased basal BG levels compared to vehicle in male (ΔBG: 31.6 ± 8.6 mg/dL vs. 11.0 ± 6.7 mg/dL, n=4, p=0.03) but not female mice. These findings establish a role for THPVN neuron PRR signaling in the pathogenesis of obesity-associated glucose metabolic impairment, and show that in male mice, basal THPVN neuron activity is required for normal glucose homeostasis. Disclosure L.Ac. souza: None. C.J.Worker: None. A.Gayban: None. M.W.Schwartz: None. Y.Feng earley: None. Funding National Institutes of Health (R01HL122770, R35HL155008, RDK83042, RDK124238)

(Pro)renin Receptor Knockdown in Tyrosine Hydroxylase neurons in the PVN Protects Against the Development of HFD‐Induced Hyperglycemia and Increases Energy Expenditure in Mice

Type 2 diabetes mellitus (T2D) is the major form of human diabetes, accounting for approximately 90–95% of diagnosed diabetes cases in the United States. The brain renin‐angiotensin system (RAS), traditionally viewed as a cardiovascular regulatory system, has recently emerged as a critical part of metabolic and energy‐expenditure signaling systems in the central nervous system (CNS). The (pro)renin receptor (PRR) is a key component of the brain RAS and plays a pivotal role in the development of hypertension. However, its importance in high fat diet (HFD)‐induced metabolic physiology is not well understood. We recently showed that neuron‐specific PRR deletion (PRRKO) improves fasting blood glucose and glucose tolerance in mice under 16 weeks of HFD. However, the specific neuronal cell types and synaptic mechanisms by which PRR acts is largely unknown. Tyrosine Hydroxylase (TH)‐containing neurons are catecholaminergic neurons found in multiple regulatory nuclei throughout the brain. The TH neurons in the paraventricular nucleus (PVN, THPVN) have been previously implicated in metabolic and cardiovascular control via inhibition of brainstem regulatory nuclei. Accordingly, we hypothesize that PRR in THPVN neurons plays a critical role in the regulation of HFD‐induced hyperglycemia. To test this hypothesis, we injected both male and female TdTomato‐PRR‐LoxP mice with adeno‐associated virus serotype 2 (AAV2)‐mediated Cre‐recombinase driven by a rat TH promoter (AAV2‐TH‐Cre) to ablate PRR in the THPVN neurons (THPVNPRRKO). The Tdtomato‐LoxP mice received the same virus were used as control. Fasting blood glucose (FBG), glucose and insulin tolerance tests were performed to assess glucose handling and development of hyperglycemia following 6‐week HFD (60% calories from fat). Mouse metabolic parameters were measured over 48 hours in Sable Promethion metabolic cages with ad libitum access to HFD. In male mice, PRR‐LoxP mice had significantly higher FBG when compared with the THPVN PRRKO mice (144.1 ± 5.74 vs. 121.9 ± 4.34 mg/dl, P= 0.0059) at the end of the six week HFD treatment. Interestingly, PRR ablation in the THPVN did not improve FBG and glucose handling in female mice. Metabolic cage measurements revealed that THPVN PRRKO mice had elevated oxygen consumption (VO2, ml/minute/g body weight) (0.047 ± 0.009 vs. 0.043 ± 0.0008 P= 0.027) and carbon dioxide emission (VCO2, ml/minute/g body weight) (0.035 ± 0.0005 vs. 0.032 ± 0.0005, P=0.0125) versus control HFD‐fed mice over 48 hours, indicating enhanced metabolic rate. The energy expenditure was significantly higher (0.0125 ± 0.00017 vs 0.0116 ± 0.00014, kCal/Hr/g body weight P=0.0026) in the THPVN PRRKO mice compared with their control counterparts. Locomotor activity over 48 hours was similar between the two groups (59.94 ± 19.6 vs. 103.5 ± 42.42 total meters traveled, P=0.204), and no significant difference in food intake (0.254 ± 0.011 vs. 0.274 ± 0.012 g, P=0.090) or body weight (42.7 ± 3.65 vs. 43.6 ± 1.41 g, P=0.842) was observed. This data suggests that PRR knockout in THPVN neurons protects against glucose dysregulation and elevates energy expenditure in mice following HFD. We conclude that PRR THPVN neurons play a key regulatory role in glucose and metabolic regulation.

Tyrosine Hydroxylase‐Positive Neurons in the Hypothalamic Paraventricular Nucleus Modulate Blood Glucose Levels in Mice

Tyrosine hydroxylase (TH)‐positive neurons in the paraventricular nucleus of the hypothalamus (PVN), termed THPVN neurons, have emerged as key metabolic regulators. Recent studies showed that THPVN neurons are mostly Gamma‐aminobutyric acid (GABA)ergic and/or dopaminergic. Previous results from our laboratory indicate that THPVN neurons project to brainstem autonomic regulatory nuclei, such as nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM). However, it is not known if THPVN neurons play a role in blood glucose regulation. We hypothesize that THPVN neurons are key regulators of blood glucose. To test our hypothesis, we used adeno‐associated virus serotype 2 (AAV2)‐mediated Cre‐recombinase driven by a rat TH promoter (AAV2‐TH‐Cre), in combination with chemogenetic approach (Designer Receptors Exclusively Activated by Designer Drugs, DREADD) to activate or inhibit THPVN neurons and continuously monitored blood glucose levels using telemetry system. hM3Dq‐LoxP (excitatory‐DREADD) and hM4Di‐LoxP (inhibitory‐DREADD) mice, 10‐14 weeks old, were bilaterally microinjected with AAV2‐TH‐Cre into the PVN. One month following AAV injection, mice were implanted with telemetry glucose transmitters (HD‐XG, DSI) to monitor blood glucose (BG) levels. To activate or inhibit THPVN neuronal activity, Clozapine‐N‐oxide (CNO, 1 mg/Kg) or vehicle were administered (i.p.) and BG was monitored by telemetry. To examine the functional role of THPVN neurons at baseline and avoid the influence of feeding on blood glucose levels, mice were fasted for 2 hours and fasting was maintained throughout the experiment. We found that activation of THPVN neurons by CNO in hM3Dq‐LoxP mice did not alter baseline blood glucose (n=5); on the other hand, inhibition of THPVN neurons by CNO in hM4Di‐LoxP mice increased baseline blood glucose levels compared with vehicle only in male mice (Male: ΔBG: 31.6 ± 8.6 mg/dL vs. 11.0 ± 6.7 mg/dL, n=4, p=0.03; Female: ΔBG: 11.0 ± 7.4 mg/dL vs. 7.3 ± 5.9 mg/dL, n=4, p=0.16). To study the functional role of THPVN neurons on whole body glucose handling, mice were fasted overnight and simultaneously injected with glucose (1 g/Kg) and either vehicle or CNO. We found that activation of THPVN neurons by CNO improved glucose clearance after glucose overload when compared with vehicle (AUC: 3133.0±109.0 vs. 6021.0±102.0, p<0.0001, n=3) in hM3Dq‐LoxP mice. In summary, our data suggest that THPVN neurons negatively regulate blood glucose possibly by their GABAergic input to downstream neural circuits. We conclude that THPVN neurons play a functional role in the regulation of glucose homeostasis; while the mechanisms remain to be determined.

A Primer for Assessing the Pathology in Mouse Models of Neuroblastoma.

Neuroblastoma, the most common extracranial solid tumor in young children, arises from the sympathetic nervous system. Our understanding of neuroblastoma has been improved by the development of both genetically engineered and xenograft mouse models of the disease. Anatomical pathology is an essential component of the phenotyping of mouse models of cancer, characterizing the morphologic effects of genetic manipulation and drug treatment. The Th-MYCN model, the most widely used of several genetically engineered mouse models of neuroblastoma, was established by targeted expression of the human MYCN gene to murine neural crest cells under the control of the rat tyrosine hydroxylase promoter. Neuroblastoma development in Th-MYCN mice is preceded by neuroblast hyperplasia-the persistence and proliferation of neural crest-derived neuroblasts within the sympathetic autonomic ganglia. The neuroblastomas that subsequently develop morphologically resemble human neuroblastoma and carry chromosomal gains and losses in regions syntenic with those observed in human tumors. In this overview, we describe the essential pathologic features for investigators when assessing mouse models of neuroblastoma. We outline human neuroblastoma as the foundation for understanding the murine disease, followed by details of the murine sympathetic ganglia from which neuroblastoma arises. Sympathetic ganglia, both with and without neuroblast hyperplasia, are described. The macroscopic and microscopic features of murine neuroblastoma are explained, including assessment of xenografts and tumors following drug treatment. An approach to experimental design is also detailed. Increased understanding of the pathology of murine neuroblastoma should improve reproducibility and comparability of research findings and assist investigators working with mouse models of neuroblastoma. © 2021 Wiley Periodicals LLC.

(Pro)Renin Receptor in the Hypothalamic Tyrosine Hydroxylase‐Containing Neurons Contributes to High Fat Diet Induced Hyperglycemia

Type 2 diabetes mellitus (T2D) is the major form of human diabetes, accounting for approximately 90–95% of diagnosed diabetes cases in the United States. The brain renin-angiotensin system (RAS), traditionally viewed as a cardiovascular regulatory system, has recently emerged as a critical part of metabolic and energy-expenditure signaling systems in the central nervous system (CNS). The (pro)renin receptor (PRR) is a key component of the brain RAS and plays a pivotal role in the development of hypertension. However, its importance in high fat diet (HFD)-induced metabolic physiology is not well understood. We recently showed that neuron-specific PRR deletion (PRRKO) improves fasting blood glucose and glucose tolerance in mice under 16 weeks of HFD. However, the specific neuronal cell types and synaptic mechanisms by which PRR acts is largely unknown. Tyrosine Hydroxylase (TH)-containing neurons are catecholaminergic neurons found in multiple regulatory nuclei throughout the brain. The TH neurons in the paraventricular nucleus (PVN, THPVN) have been previously implicated in metabolic and cardiovascular control via inhibition of brainstem regulatory nuclei. Accordingly, we hypothesize that PRR in THPVN neurons plays a critical role in the regulation of HFD-induced hyperglycemia. To test this hypothesis we injected male PRR-LoxP mice with adeno-associated virus serotype 2 (AAV2)-mediated Cre-recombinase driven by a rat TH promoter (AAV2-TH-Cre) to ablate PRR in the THPVN neurons (THPVN PRRKO) or AAV2-eGFP (Control) prior to 6 week HFD (60% calories from fat) treatment. Fasting blood glucose (FBG), glucose and insulin tolerance tests were performed to assess glucose handling and development of hyperglycemia following HFD. PRR-LoxP mice had significantly higher FBG when compared with the THPVN PRRKO mice (139.3 ± 6.551 vs. 118.0 ±3.715 mg/dl, P= 0.0409) and trended towards improved glucose tolerance when compared with the control mice following the treatment period (Area under the curve-AUC: 12990 ± 1494 vs. 9035 ± 1354, P=0.0782), suggesting that THPVN PRRKO protects against hyperglycemia and may improve glucose tolerance after 6 weeks HFD treatment. No significant difference between the two groups was observed in the insulin tolerance test (AUC: 10833 ± 727.7 vs. 10135 ± 729.5, P=0.7142), suggesting a possible mechanism of THPVN PRR glycemia regulation independent of insulin resistance. These data indicate a critical role of THPVN PRR in the regulation of HFD-induced hyperglycemia and a potential pathway for understanding the mechanism behind the metabolic action of neuronal PRR.

Tyrosine Hydroxylase‐Positive Neurons in the Hypothalamic Paraventricular Nucleus Modulate Glucose Homeostasis in Mice

Tyrosine hydroxylase (TH)-positive neurons in the paraventricular nucleus of the hypothalamus (PVN), termed THPVN neurons, have emerged as key metabolic regulators. Recent studies showed that THPVN neurons are mostly Gamma-aminobutyric acid (GABA)ergic and/or dopaminergic. However, it is not known if THPVN neurons play a role in blood glucose regulation. We hypothesize that THPVN neurons are key regulators of blood glucose. To test our hypothesis, we used adeno-associated virus serotype 2 (AAV2)-mediated Cre-recombinase driven by a rat TH promoter (AAV2-TH-Cre), in combination with chemogenetic approach (Designer Receptors Exclusively Activated by Designer Drugs, DREADD) to activate or inhibit THPVN neurons and continuously monitored blood glucose levels using telemetry system. The hM3Dq-LoxP (excitatory-DREADD) and hM4Di-LoxP (inhibitory-DREADD) mice, 10-14 weeks old, were bilaterally microinjected with AAV2-TH-Cre into the PVN. One month after AAV injection, mice were implanted with telemetry glucose transmitters (HD-XG, DSI) to monitor blood glucose (BG) levels. To activate or inhibit the THPVN neuronal activity, Clozapine-N-oxide (CNO, 3 mg/Kg) or vehicle were administered (i.p.) and BG was monitored by telemetry. We found that activation of THPVN neurons by CNO in hM3Dq-LoxP mice decreased baseline blood glucose compared with the vehicle (ΔBG: -9.8 ± 0.2 mg/dL vs. 0.5 ± 0.1 mg/dL, n=5, p<0.0001); on the other hand, inhibition of THPVN neurons by CNO in the hM4Di-LoxP mice increased baseline blood glucose levels compared with the vehicle (ΔBG: 12.2 ± 0.2 mg/dL vs. 0.6 ± 0.3 mg/dL, n=3, p<0.0001). To exam the functional role of THPVN neurons on whole body glucose handling, mice were fasted overnight and simultaneously injected with glucose (1 g/Kg) and either CNO or vehicle. We found that activation of THPVN neurons by CNO improved glucose clearance after the glucose loading compared with vehicle (AUC: 3028.0±53.0 vs. 6021.0±102.0, p<0.001, n=3) in hM3Dq-LoxP mice. In summary, our data suggest that THPVN neurons negatively regulate blood glucose possibly by their GABAergic input to downstream neural circuits. We conclude that THPVN neurons play a functional role in the regulation of glucose homeostasis; while the mechanisms remain to be determined.

Characterization of Tyrosine Hydroxylase‐Positive Neurons Projections from the Paraventricular Nucleus of Hypothalamus to Hindbrain Autonomic Regulatory Centers

Hypothalamus is a key brain region for cardiovascular, behavioral, and metabolic regulation that contains complex type of excitatory or inhibitory neurons. Tyrosine hydroxylase (TH)-positive neurons in the hypothalamus play an important role in cardiometabolic regulation; however, the anatomical projections of TH neurons in the paraventricular nucleus hypothalamus (PVN) to key parasympathetic and sympathetic premotor nuclei in preautonomic regions of the brain and their connections to the liver remains undefined. This study aims to characterize the projections from THPVN neurons to ventral preautonomic regulating centers and their connection to liver neuronal projections. To achieve our goal, Cre recombinase-dependent TIT2L-eGFP reporter mice were microinjected with adeno-associated virus encoding the Cre recombinase driven by a rat TH promoter (AAV2-TH-Cre, 6.8 x 108 vg/site) bilaterally into the PVN using a stereotaxic apparatus. A retrograde pseudorabies virus with red fluorescence reporter (PRV-614-RFP) was injected into the liver (3 sites, 3ul/site) to identify liver-related neurons in the brainstem preautonotmic centers. One month after injection, mice were transcardially perfused and brain tissues were processed for confocal imaging. The AAV2-TH-Cre induced a robust expression of GFP mostly in the PVN neuronal cell bodies but not in other brain regions. In the brainstem preautonomic centers, we found dense projections in the nucleus tractus solitarius (NTS) and some projections to the dorsal motor nucleus of the vagus (DMV) and rostral ventrolateral medulla (RVLM). Interestingly, there was also dense projects into the median eminence (ME) that regulates neural hormones release. Importantly, the PRV-RFP retrograded from liver was found throughout the DMV and ventrolateral NTS . Particularly in the NTS, there was dense synaptic contact of the THPVN neuronal projections with the liver related neurons (PRV-RFP+ cell). In addition, we also observed that some PRV-RFP+ cells colocalized with TH-positive neurons in the PVN. In sum, the THPVN neurons project anatomically to the autonomic and neurosecretory regions of brain and may modulate the cardiometabolic activity and liver metabolism through these projections.

A quantitative evaluation of a 2.5-kb rat tyrosine hydroxylase promoter to target expression in ventral mesencephalic dopamine neurons in vivo

Adeno-associated viruses (AAVs) have become powerful tools in neuroscience for both basic research and potential therapeutic use. They have become especially important tools for optogenetic experiments based on their ability to achieve transgene expression in postmitotic neurons with regional selectivity. With the use of appropriate promoter elements they can achieve cellular specificity as well. One population of neurons that plays a central role in human neurodegenerative and psychiatric diseases are the dopamine neurons of the midbrain. To study these neurons in vivo with advanced techniques it would be highly advantageous to characterize an appropriate specific promoter. To this end we have characterized a 2.5-kb sequence of the rat tyrosine hydroxylase (TH) promoter. The rTHp(2.5) promoter induced expression of the fluorescent reporter protein mCherry in SN dopamine neurons. Although it showed excellent specificity in cortex and striatum, where no reporter expression was observed, in the SN region many neurons expressed reporter but not TH. We show that some of the TH negativity is due to the suppression of its expression by the transgene. We conclude that rTHp(2.5) does preferentially label dopamine neurons but its specificity is not complete within the substantia nigra and caution must be used.

Opposite effects of acute and chronic amphetamine on Nurr1 and NF-κB p65 in the rat ventral tegmental area

Dopamine neurons are overstimulated by drugs of abuse and suffer molecular alterations that lead to addiction behavior. Nurr1 is a transcription factor crucial for dopamine neurons survival and dopamine production, activating the transcription of key genes like tyrosine hydroxylase (TH). Interestingly, nuclear factor-kappa B (NF-κB) has emerged as a new Nurr1 partner in response to inflammatory stimulus. In this study we evaluated the effects of single and repeated amphetamine administration in the expression of Nurr1 and the NF-κB p65 subunit in the rat ventral tegmental area (VTA). We found that acute amphetamine treatment increased Nurr1, p65 and TH protein levels in the VTA. On the other hand, chronic amphetamine treatment decreased Nurr1 and p65 protein levels, but TH was unchanged. Mammalian reporter assays in cell lines showed that p65 represses Nurr1 transcriptional activity in an artificial promoter driven by Nurr1 response elements and in the native rat TH promoter. These results indicate that Nurr1 and NF-κB p65 factors are involved in the adaptive response of dopamine neurons to psychostimulants and that both transcription factors could be regulating Nurr1-dependent transactivation in the VTA.

A 16 bp upstream sequence from the rat tyrosine hydroxylase promoter supports long-term expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system

Helper virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100bp fragments that each support long-term expression, and are contained within an ~320bp fragment located ~3kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100bp fragments contained 44 or 23bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42bp sequence, centered on the 23bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16bp maximum essential sequence. This 16bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.

Adenosine A2A receptor but not HIF‐1 mediates Tyrosine hydroxylase induction in hypoxic PC12 cells

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines released by oxygen-sensitive cells in response to hypoxic conditions. Adenosine is released in response to hypoxia in the central nervous system and CGS21680, an adenosine A(2)A receptor agonist, induces TH transcription. As we have previously demonstrated the A(2)A receptor-mediated induction of HIF-1 in macrophages and hepatocytes, we investigated the involvement of HIF-1 in the adenosine-mediated activation of TH expression. Exposure to adenosine or CGS21680 increased TH mRNA and protein levels in PC12 cells. Transcription of a reporter gene under the control of the wild type rat TH promoter was induced 3.5-fold in CGS21680-treated cells, but neither the mutation of the hypoxia responsive element in the TH promoter nor the co-transfection of a dominant negative of the HIF-1 beta subunit prevented the increase in transcription; furthermore, CGS21680 increased CREB binding activity but did not induce HIF-1 DNA binding activity or protein levels. To investigate whether HIF-1 was involved in the hypoxia-mediated induction of TH, PC12 cells were exposed to hypoxia in the presence of the A(2)A receptor antagonist ZM241385, which prevented hypoxia-dependent TH induction despite HIF-1 activation; in line with this finding, the inhibition of HIF-1 did not abolish TH induction in hypoxic PC12 cells. These results indicate that, under hypoxic conditions, TH (a key factor in systemic adaptation to reduced oxygen availability) is not regulated by HIF-1, the primary modulator of the response to hypoxia, but by the adenosine A(2)A receptor-mediated signalling pathway.

A Simple PCR Method for Rapid Genotype Analysis of the TH-MYCN Transgenic Mouse

BackgroundThe TH-MYCN transgenic mouse is the most widely used murine model of human neuroblastoma, in which a human MYCN oncogene is targeted to neuroectodermal cells of developing mice under the influence of the rat tyrosine hydroxylase promoter. So far, homozygous transgenic mice have been identified by either Southern blot or quantitative real-time PCR.Principal FindingsTo establish a simple and reliable genotyping method by conventional PCR, we confirmed the integration of the transgene in the TH-MYCN transgenic mouse by Southern blot and inverse PCR analyses. Our results showed that either five or six copies were found to be inserted in a head-to-tail tandem configuration at a single locus. The MYCN transgene/host DNA junction was sequenced and the integration site was identified at chromosome 18qE4. Finally, we succeeded in designing rapid, simple and reliable genotyping method by common PCR using primers flanking the integrated TH-MYCN transgene.ConclusionWe established a simple and reliable genotyping PCR method for determining the integration site of the TH-MYCN transgene that enables all possible genotypes to be distinguished within several hours. TH-MYCN mice are excellent model for human neuroblastoma study, thus our results will largely be useful for facilitating the pace of neuroblastoma study, including in the study of the tumourigenic process, and in the development of therapies to treat patients suffering from neuroblastoma.

Intravenous glial‐derived neurotrophic factor gene therapy of experimental Parkinson's disease with Trojan horse liposomes and a tyrosine hydroxylase promoter

Rats with experimental Parkinson's disease (PD) are treated with intravenous glial-derived neurotrophic factor (GDNF) plasmid DNA, non-viral gene therapy using Trojan horse liposomes (THLs) targeted with a monoclonal antibody (MAb) to the rat transferrin receptor (TfR). Expression of the transgene is confined to catecholaminergic cells by placement of the GDNF gene under the influence of the rat tyrosine hydroxylase (TH) promoter. A 13-kb eukaryotic expression plasmid, designated pTHpro-GDNF, is engineered in which the human prepro GDNF cDNA is driven by 8 kb of the 5'-flanking sequence of the rat TH promoter (pro), and is 3'-flanked by the bovine growth hormone transcription termination sequence. The pTHpro-GDNF plasmid DNA is encapsulated in THLs targeted with a TfRMAb, and a single intravenous injection is given to rats at 2 weeks after experimental PD is induced by intra-cerebral 6-hydroxydopamine. Expression of the GDNF gene, under the influence of the TH promoter, is restricted compared to GDNF expression under the influence of the cytomegalovirus promoter. GDNF is elevated only in organs of the rat where TH gene expression is observed, including the substantia nigra, liver and adrenal gland. The single, delayed intravenous administration of the GDNF gene therapy causes a lasting reduction in apormorphine-induced rotation, which is correlated with a 19-fold increase in striatal TH enzyme activity. Both dose-response and time-responses are observed. Sustained therapeutic effects are achieved in experimental PD with a delayed single intravenous dosing of GDNF plasmid DNA gene therapy, using receptor-targeted THLs and a region-specific promoter.

GDNF fusion protein for targeted‐drug delivery across the human blood–brain barrier

Glial-derived neurotrophic factor (GDNF) is a neurotrophin that could be developed as a neurotherapeutic for Parkinson's disease, stroke, and motor neuron disease. However, GDNF does not cross the blood-brain barrier (BBB). Human GDNF was re-engineered by fusion of the mature GDNF protein to the carboxyl terminus of the chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb-GDNF fusion protein is bi-functional, and both binds the HIR, to trigger receptor-mediated transport across the BBB, and binds the GDNF receptor (GFR)-alpha1, to activate GDNF neuroprotection pathways behind the BBB. COS cells were dual transfected with the heavy chain (HC) and light chain fusion protein expression plasmids, and the HC of the fusion protein was immunoreactive with antibodies to both human IgG and GDNF. The HIRMAb-GDNF fusion protein bound with high affinity to the extracellular domain of both the HIR, ED(50) = 0.87 +/- 0.13 nM, and the GFRalpha1, ED(50) = 1.68 +/- 0.17 nM. The HIRMAb-GDNF fusion protein activated luciferase gene expression in human neural SK-N-MC cells dual transfected with the c-ret kinase and a luciferase reporter gene under the influence of the rat tyrosine hydroxylase promoter, and the ED(50), 1.68 +/- 0.45 nM, was identical to the ED(50) in the GFRalpha1 binding assay. The fusion protein was active in vivo in a rat middle cerebral artery occlusion model, where the stroke volume was reduced 77% (P < 0.001). In conclusion, these studies describe the re-engineering of GDNF, to make this neurotrophin transportable across the human BBB.

The Effect of Truncated Human α-Synuclein (1–120) on Dopaminergic Cells in a Transgenic Mouse Model of Parkinson's Disease

Alpha-Synuclein is thought to play an important role in the pathology of Parkinson's disease (PD). Truncated forms of this protein can be found in PD brain extracts, and these species aggregate faster and are more susceptible to oxidative stress than the full-length protein. We investigated the effect of truncated alpha-synuclein on dopaminergic cells using a transgenic mouse expressing alpha-synuclein (1-120) driven by the rat tyrosine hydroxylase promoter on a mouse alpha-synuclein null background. We found a selective reduction in the yield of dopaminergic cells from transgenic embryonic ventral mesencephalic cell cultures. However, in vivo the substantia nigra/ventral tegmentum dopaminergic cell counts were not reduced in transgenics, although these mice are known to have reduced striatal dopamine. When transplanted to the striatum in the unilateral 6-hydroxydopamine-lesioned mouse model of PD, dopaminergic cells derived from transgenic embryonic ventral mesencephala were significantly smaller at 6 weeks, and showed a trend towards being less effective at ameliorating rotational asymmetry than those from control alpha-synuclein null mice. These results suggest that alpha-synuclein (1-120) renders dopaminergic cells more susceptible to stress, which may have important implications as to how this truncated protein might contribute to dopaminergic cell death in sporadic PD.

Accumulation of phosphorylated α‐synuclein in dopaminergic neurons of transgenic mice that express human α‐synuclein

Parkinson's disease is neuropathologically characterized by the presence of Lewy bodies, whose major component is alpha-synuclein. We had previously generated transgenic mice that expressed human alpha-synuclein carrying an Ala53Thr point mutation (halpha-syn140m) under the control of the rat tyrosine hydroxylase promoter and found that halpha-syn140m was localized not only in the cytoplasm but also in the nuclei of mesencephalic dopaminergic neurons. In the present study, we carried out immunohistochemical analysis of the brain of Tg mice using anti-PSer129, an antibody that specifically recognizes alpha-synuclein phosphorylated at Ser129. The antibody detected only phosphorylated halpha-syn140m, whereas phosphorylation of endogenous alpha-synuclein, if any, was below the detection limit of the method employed. The analysis showed that approximately one-third of the halpha-syn140m-positive neurons in the midbrain of heterozygous Tg mice were concomitantly reactive to anti-PSer129. The ratio almost doubled in homozygotes, indicating that the phosphorylation level depends directly on the amount of substrate. In addition, the ratio did not change at least up to 48 weeks of age. These data strongly suggest that halpha-syn140m underwent constitutive phosphorylation and that the phosphorylation level was maintained to a certain level until the aged stages. Remarkably, halpha-syn140m localized in the nuclei seemed to be preferentially phosphorylated compared with that in the cytoplasm. Among kinases that have been reported to be involved in the phosphorylation of alpha-synuclein, the beta subunit of casein kinase-2 was detected in the nuclei by immunohistochemistry. These data imply that at least casein kinase-2 is involved in the phosphorylation of halpha-syn140m in the Tg mice.

Isolation of an enhancer from the rat tyrosine hydroxylase promoter that supports long-term, neuronal-specific expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system

Direct gene transfer into neurons, using a virus vector, has been used to study neuronal physiology and learning, and has potential for supporting gene therapy treatments for specific neurological diseases. Many of these applications require high-level, long-term recombinant gene expression, in forebrain neurons. We previously showed that addition of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a neurofilament heavy gene (NF-H) promoter supports long-term expression in forebrain neurons, from helper virus-free Herpes Simplex Virus (HSV-1) vectors. This element in the TH promoter satisfied the definition of an enhancer; it displayed activity at a distance from the basal promoter, and in both orientations. This enhancer supported physiological studies that required long-term expression; a modified neurofilament promoter, containing an insulator upstream of the TH-NFH promoter, supported expression in ∼ 11,400 striatal neurons at 6 months after gene transfer, and expression for 7, 8, or 14 months, the longest times tested. In contrast, the NF-H promoter alone does not support long-term expression, indicating that the critical sequences are in the 6.3 kb fragment of the TH promoter. In this study, we performed a deletion analysis to identify the critical sequences in the TH promoter that support long-term expression. We localized these critical sequences to an ∼ 320 bp fragment, and two subfragments of ∼ 100 bp each. Vectors that contained each of these small fragments supported levels of long-term, neuronal-specific expression that were similar to the levels supported by a vector that contained the initial 6.3 kb fragment of the TH promoter. These small fragments of the TH promoter may benefit construction of vectors for physiological studies, and may support studies on the mechanism by which this enhancer supports long-term expression.

Pathological Changes in Dopaminergic Nerve Cells of the Substantia Nigra and Olfactory Bulb in Mice Transgenic for Truncated Human α-Synuclein(1–120): Implications for Lewy Body Disorders

Dysfunction of the 140 aa protein alpha-synuclein plays a central role in Lewy body disorders, including Parkinson's disease, as well as in multiple system atrophy. Here, we show that the expression of truncated human alpha-synuclein(1-120), driven by the rat tyrosine hydroxylase promoter on a mouse alpha-synuclein null background, leads to the formation of pathological inclusions in the substantia nigra and olfactory bulb and to a reduction in striatal dopamine levels. At the behavioral level, the transgenic mice showed a progressive reduction in spontaneous locomotion and an increased response to amphetamine. These findings suggest that the C-terminal of alpha-synuclein is an important regulator of aggregation in vivo and will help to understand the mechanisms underlying the pathogenesis of Lewy body disorders and multiple system atrophy.

Prolonged Activation of cAMP-response Element-binding Protein and ATF-2 Needed for Nicotine-triggered Elevation of Tyrosine Hydroxylase Gene Transcription in PC12 Cells

Phosphorylation (P-) of cAMP-response element-binding protein (CREB) by protein kinase A or mitogen-activated protein kinases was implicated in mediating the increased tyrosine hydroxylase (TH) gene expression after prolonged exposure to nicotine in vivo and in cell culture. We examined the time course and signaling pathways for phosphorylation of CREB and possible involvement of ATF-2. Treatment of PC12 cells with 200 microm nicotine triggered rapid but transient elevation of P-CREB followed by a second sustained rise after 2-5 h of continuous nicotine. In contrast, ERK1/2 was only phosphorylated with short term nicotine exposure. MEK inhibitor U0126 abolished nicotine-induced rise in P-ERK1/2, but not P-CREB, nor did it inhibit nicotine-evoked elevation in TH promoter activity, indicating that ERK1/2 was not needed for induction of TH gene expression by nicotine. In contrast, protein kinase A inhibitor H-89 or Ca(2+)/calmodulin-activated protein kinase inhibitor KN-93 reduced the nicotine-triggered rise in P-CREB and TH promoter activity. There was a delayed elevation of P-ATF-2 after 1 h of nicotine treatment, accompanied by increased ATF-2 protein. Upstream kinase JNK, but not p38, was phosphorylated especially after 5 min to 2 h of nicotine exposure. To examine the requirement for CREB and ATF-2, cells were transfected with dominant negative forms of ATF-2 or CREB. Both reduced the basal TH promoter activity and the response to nicotine. Knockdown of ATF-2 or CREB with siRNA did not alter basal TH promoter activity or mRNA but greatly attenuated the response to nicotine. The results suggest that both ATF-2 and CREB mediate activation of TH gene transcription by nicotine.

Bcl-x is required for proper development of the mouse substantia nigra.

Recent findings have uncovered a role for the Bcl-x gene in the survival of dopaminergic neurons. The exact nature of this role has been difficult to examine because of the embryonic lethality of Bcl-x gene disruption in mouse models. Here we report the generation catecholaminergic cell-specific conditional Bcl-x gene knock-out mice using Cre-lox recombination technology. First we produced transgenic mice that express Cre recombinase from an exogenous rat tyrosine hydroxylase promoter (TH-Cre mice). These mice were crossed to Z/AP and Z/EG reporter mouse strains to verify catecholaminergic (TH-positive) cell-specific Cre expression. The TH-Cre mice then were mated to mice possessing the Bcl-x gene flanked by loxP sites, thereby producing offspring with Bcl-x deletion limited to catecholaminergic cells. The resulting mice are viable but have one-third fewer catecholaminergic neurons than do control animals. They demonstrate a deficiency in striatal dopamine and also tend to be smaller and have decreased brain mass when compared with controls. Surprisingly, surviving neurons were found that lacked Bcl-x immunoreactivity, thereby demonstrating that this gene is dispensable for the ongoing survival of a subpopulation of catecholaminergic cells.