Prokineticin-2 Upregulates GDNF in Astrocytes and Pharmacological Modulation of PK2 Receptors offers Neuroprotection in Experimental Models of Parkinson’s Disease

Prokineticin-2 Upregulates GDNF in Astrocytes and Pharmacological Modulation of PK2 Receptors offers Neuroprotection in Experimental Models of Parkinson’s Disease | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Prokineticin-2 Upregulates GDNF in Astrocytes and Pharmacological Modulation of PK2 Receptors offers Neuroprotection in Experimental Models of Parkinson’s Disease Jie Luo , View ORCID Profile Griffin Clabaugh , Matthew Neal , Minhong Huang , Souvarish Sarkar , Gary Zenitsky , Huajun Jin , Vellareddy Anantharam , Canan G. Nebigil , Laurent Désaubry , Arthi Kanthasamy , Anumantha G. Kanthasamy doi: https://doi.org/10.1101/2025.09.08.674934 Jie Luo 1 Department of Biomedical Sciences, Iowa State University , Ames, IA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Griffin Clabaugh 2 Center for Neurological Disease Research, Department of Physiology and Pharmacology, University of Georgia , Athens, GA 3 Department of Biochemistry and Molecular Biology, University of Georgia , Athens, GA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Griffin Clabaugh Matthew Neal 1 Department of Biomedical Sciences, Iowa State University , Ames, IA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Minhong Huang 1 Department of Biomedical Sciences, Iowa State University , Ames, IA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Souvarish Sarkar 1 Department of Biomedical Sciences, Iowa State University , Ames, IA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gary Zenitsky 2 Center for Neurological Disease Research, Department of Physiology and Pharmacology, University of Georgia , Athens, GA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Huajun Jin 2 Center for Neurological Disease Research, Department of Physiology and Pharmacology, University of Georgia , Athens, GA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Vellareddy Anantharam 2 Center for Neurological Disease Research, Department of Physiology and Pharmacology, University of Georgia , Athens, GA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Canan G. Nebigil 4 Regenerative Nanomedicine (UMR 1260), INSERM, University of Strasbourg, Center of Research in Biomedicine of Strasbourg , Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Laurent Désaubry 4 Regenerative Nanomedicine (UMR 1260), INSERM, University of Strasbourg, Center of Research in Biomedicine of Strasbourg , Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Arthi Kanthasamy 2 Center for Neurological Disease Research, Department of Physiology and Pharmacology, University of Georgia , Athens, GA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anumantha G. Kanthasamy 1 Department of Biomedical Sciences, Iowa State University , Ames, IA 2 Center for Neurological Disease Research, Department of Physiology and Pharmacology, University of Georgia , Athens, GA 3 Department of Biochemistry and Molecular Biology, University of Georgia , Athens, GA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: anumantha.kanthasamy{at}uga.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Despite a wealth of preclinical studies establishing neuroprotective and neurorestorative properties of glial cell-line-derived neurotrophic factor (GDNF) in animal models of Parkinson’s disease (PD), clinical trials utilizing direct intracranial infusion of GDNF protein, or adeno-associated virus (AAV)-mediated GDNF gene transfer has not achieved the desired efficacy, largely due to challenges in delivery methods. Given GDNF’s strong potential for neuroprotection, alternative strategies to elevate its expression by beyond invasive injection or genetic manipulation remain a promising therapeutic avenue for PD. We previously reported that prokineticin signaling provides a compensatory protective response against dopaminergic neuronal degeneration in cell and animal models of PD. Herein, we report a novel finding that PK2 regulates GDNF gene expression in astrocytes, suggesting that PK2 signaling can be harnessed for neuroprotection in PD. Treatment of cultured astrocytes with the PK2 protein, PK2 gene overexpression or prokineticin receptor 1 (PKR1) agonist IS20 significantly induced the GDNF gene expression and the protein secretion, resulting in enhanced dopaminergic cell survival in cell culture models of PD. Importantly, systemic administration of IS20 through intraperitoneal or intranasal routes elevated GDNF levels in the mouse brain, including the nigrostriatal system. Furthermore, IS20 treatment conferred significant neuroprotective effects in both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced and MitoPark transgenic mouse models of PD. Collectively, our translational findings suggest that pharmacological modulation PK2 signaling may unlock the full clinical benefit of GDNF, offering a novel and non-invasive therapeutic strategy for Parkinson’s disease. Introduction As the second most common neurodegenerative disease, Parkinson’s disease (PD) affects around 1% of the world’s population ≥65 years old. PD is mainly characterized by progressive degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc) in the ventral midbrain, resulting in dopamine depletion within the striatum. This neurodegeneration leads to hallmark motor symptoms, including bradykinesia, rigidity, and tremor 1 , 2 . Notably, autonomic and other non-motor deficits such as sleep disturbances, anosmia, gastrointestinal dysfunction, and mood disorders often precede the onset of motor symptoms, suggesting early involvement of extranigral brain regions in PD pathogenesis. The DA precursor, 3,4-dihydroxy-L-phenylalanine (L-DOPA), is the current standard-of-care treatment strategy for PD, which temporarily alleviates motor symptoms by increasing brain dopamine levels. However, L-DOPA has limited efficacy in addressing motor symptoms and does not halt disease progression 3 – 5 . Since no disease-modifying therapy has been developed, the progressive nature of the disease demands dosage titration involving increasingly higher doses of L-DOPA, resulting in an array of motor fluctuations and other severe complications, including dyskinesia 2 , 6 . Glial cell-line-derived neurotrophic factor (GDNF) is well characterized neurotrophic factor involved in the development and maintenance of multiple neuronal systems including those of the kidney, enteric nervous system (ENS), and central nervous system (CNS) 7 – 10 . Since its discovery in 1992, GDNF has been recognized as one of the most potent neuroprotective agents, particularly within in the DAergic system 11 , 12 . DAergic neurons express both the GDNF family receptor GFRα1 and GDNF co-receptor RET, through which GDNF promotes neuronal survival, regeneration and functional maintenance in the SN DAergic neurons. GDNF also increases DA content and uptake, stimulates neurite extension, and increases tyrosine hydroxylase (TH) in cell culture models 12 , 13 . In vivo evidence further supports that GDNF is necessary for the homeostatic maintenance of DAergic neurons during later stages of development and throughout adulthood. Supporting this notion, conditional knockout (cKO) of GDNF in mice starting at 1-mo-old results in the selective decrease of TH immunoreactivity and a 60-70% loss of DA midbrain neurons by seven months of age, underscoring GDNF’s indispensable role in DA neuron survival under physiological conditions 10 . Numerous preclinical studies using rodent and non-human primate models of PD have demonstrated the therapeutic potential of GDNF 14 – 17 . However, early clinical trials delivering recombinant (r)GDNF protein using intracranial administration failed to uniformly diffuse into deeper layers of the brain and did not slow down neurodegeneration or improve motor function 18 . While improvements in GDNF delivery have achieved moderate efficacy 19 , the difficult challenge of long-term GDNF delivery into the brain remains. Additional safety concerns including cerebellar toxicity were also evident 20 . Gene therapy approaches using adeno-associated virus (AAV)-mediated GDNF gene delivery offered the potential to overcome the delivery limitations and were deemed safe in a Phase I clinical trials. Nevertheless, Phase II trials failed to demonstrate therapeutic efficacy 21 – 23 . Moreover, AAV-mediated gene delivery is inherently less controllable than conventional pharmacotherapies due to irreversible integration into the host genome, raising concerns about long-term safety and regulation 24 . Failures of current GDNF trials underscore the critical need to elucidate events upstream of the GDNF signaling pathway and to disentangle the complex signaling pathways in crosstalk with GDNF. Controlling endogenous GDNF expression would constitute a powerful target in neuropharmacology and warrants increased translational research efforts 25 , 26 . Setbacks of current AAV-mediated DAergic GDNF gene delivery trials force the rethinking of alternative strategies, including expressing GDNF in striatal astrocytes. Astrocytes secrete the neurotrophic factors NGF, BDNF, and GDNF 27 , and are considered primary source of GDNF in the brains of PD patients 28 , 29 . The role of astrocytes in maintaining a healthy environment for proper neuron function has been increasingly recognized, yet their potential as a source of nigral GNDF and as a viable therapeutic target remains underexplored. Harnessing astrocyte-mediated endogenous GDNF expression could offer a more physiologically relevant and controllable approach to neuroprotection in PD, warranting further investigation in translational research. Initially identified from a nontoxic component of black mamba snake venom 30 , the prokineticin family proteins have been shown to interact with the GDNF signaling pathway in the peripheral nervous system (PNS) during development of the ENS 7 , 8 , 31 , 32 . Disruptions in either signaling pathway are implicated in Hirschsprung disease, a rare congenital disorder of the ENS 33 , highlighting the functional interdependence of GDNF and prokineticin signaling. The well-established crosstalk between these pathways in the ENS raises the intriguing possibility that the prokineticin signaling pathway also engages in similar crosstalk with GDNF in the CNS. Interestingly, both GDNF and prokineticin-2 (PK2) have been identified as potent chemoattractants for axons during neurogenesis/neuritogenesis. PK2, in particular, is essential for the complete development of olfactory bulb (OB) 34 . Importantly, our previous work demonstrated that PK2 upregulation mediates a protective, compensatory response during neurodegeneration by activating the AKT and ERK pathways and enhancing mitochondrial biogenesis 35 . AAV2/5-mediated delivery of PK2 conferred neuroprotection against Parkinsonian toxicants in the SN and striatum 35 . However, evidence is lacking for a fundamental relationship between PK2/PKR1 and GDNF/RET signaling in the CNS and the nigrostriatal system. Intriguingly, PKR1 is mainly expressed in astrocytes 36 , raising the possibility that GDNF may be co-upregulated with the prokineticin pathway in astrocytes. The preferential expression of PKR1 over PKR2 in astrocytes further supports the feasibility of selectively targeting astrocytes using PKR1-specific agonists. Based on this therapeutic rationale, using IS20, a lipophilic small molecule receptor agonist specific to PKR1 37 , we hypothesized that positive modulation of the expression and secretion of GDNF in astrocytes of the nigrostriatal system could be achieved via pharmacological activation of PK2/PKR1 signaling. This astrocyte-derived trophic support may, in turn, promote the DAergic neuronal survival and exert both neuroprotective and neurorestorative effects in experimental models of PD. In this study, we report that 1) PK2/PKR1 signaling represents a novel, druggable pathway for modulating GDNF expression in the CNS, particularly in astrocytes, 2) the upregulation of GDNF through PK2/PKR1 signaling can be pharmacologically activated by intranasal (IN) or intraperitoneal delivery of the PKR1 receptor agonist IS20, and 3) IS20 could rescue DA neurons and neuronal projections from neurodegeneration in genetic and chemically-induced mouse models of PD. Unlike the uncontrolled, long-term continuous expression driven by intracerebral injections of GDNF viral vectors, which leads to unwanted compensatory responses 38 – 40 , systemic administration small molecule IS20 showed no such adverse effects, nor any overt toxicity in mice. This distinction highlights advantage of pharmacologically modulating endogenous GDNF expression more controlled and physiologically relevant manner. Thus, by pharmacologically modulating the magnitude and duration of endogenous GDNF expression using small-molecule IS20 treatment, GDNF’s established clinical benefits could be fully harnessed. Results Prokineticin receptor 1 is activated by rPK2 protein and receptor agonist IS20 Previous work in our lab has demonstrated that PK2 signaling functions as a potent neuroprotective response during early stages of DAergic neurodegeneration in models of PD. Given the established crosstalk between prokineticin signaling and GDNF during development, and the well-characterized protective effect of GDNF in preclinical models of PD, we hypothesized that PK2’s neuroprotective functionalities are afforded through activation of GDNF signaling. PKR1 is expressed on the membrane of DAergic neurons and Type 1 astrocytes in the nigrostriatal system. We confirmed PKR1 and GDNF expression in the substantia nigra by immunostaining human postmortem nigral sections probing for PKR1 and GDNF (Supplementary Fig. 1a). Interestingly, we found that PKR1 is highly expressed in the SN and colocalized with GDNF, indicating the possibility of GDNF expression being downstream of PKR1 signaling. We also found a marked decrease in GDNF and mRNA (Supplementary Fig. 1b) in PD patient nigral lysates compared to control samples in line with some previous reports 29 , 41 while conflicting with others 42 . This decrease in GDNF highlights the lack of trophic support in the nigrostriatal system in PD patients and the therapeutic potential of restoring GDNF levels in the SN. To examine whether the PK2 agonist IS20 can positively modulate GDNF expression, our initial set of experiments was conducted to confirm that IS20 activates PKR1 as has been previously reported 37 , 43 . Once activated, PKR1 initiates downstream signaling that culminates in the rise of Ca 2+ in the cytosol 37 , 44 . In silico modeling with AlphaFold3 of PK2 binding to PKR1 confirms that the AVITGA sequence motif resides in the binding pocket of PKR1 (Supplementary Fig. 2a). This is in alignment with previous reports of AVITGA being required for receptor binding. In silico analysis also revealed that IS20 can bind to PKR1 with high affinity (Supplementary Fig. 2b). To examine PKR1 activation in vitro , we used a CHO cell line stably expressing PKR1. The PKR1-overexpressing cells were treated with 100 nM rPK2 or 10 µM IS20, and PKR1 activation was monitored by measuring intracellular Ca 2+ mobilization in real time over 180 s using a Fluo-4 NW calcium assay kit. Upon treatment with rPK2, intracellular Ca 2+ concentration rose rapidly, peaking at 20 s post-treatment (Supplementary Fig. 2c), indicative of a Gαq1 mechanism for PKR1 activation. Treatment with 10 µM of IS20 caused Ca 2+ concentration to rise more slowly, peaking at 180 s post-treatment (Supplementary Fig. 2d), suggesting that IS20 induces a similarly large but relatively delayed activation of PKR1 compared to rPK2. Recombinant PK2 and PK2 agonist IS20 upregulate GDNF expression in human astrocytes To examine the effects of PK2 signaling activation on GDNF expression in astrocytes, we first incubated U373 human astrocyte cells with rPK2 protein dissolved in reduced serum (2% FBS). Treatment with 25 nM rPK2 for 8 h induced GDNF mRNA expression levels to nearly 2.5-fold above control ( Fig. 1a ), and treatment for 10 h induced GDNF protein levels 1.7-fold above control ( Fig. 1b ). The rPK2-induced upregulation of GDNF mRNA was completely attenuated by co-treatment with the prokineticin receptor antagonist PC-7 ( Fig. 1a ). To validate these findings in human cells, primary human astrocytes were incubated with 25 nM rPK2 for 4, 8, and 12 h. We found that rPK2 incubation elicited a time-dependent increase in GDNF mRNA over 8 h that diminished by 12 h ( Fig. 1c ). Immunocytochemistry (ICC) analysis confirmed the increase in GDNF protein levels compared to control ( Fig. 1d ). Download figure Open in new tab Figure 1. PK2 and PK2 agonist IS20 induce GDNF expression in human astrocytes. (a) qPCR of GDNF gene expression in U373 human astroglioma cell line treated with PK2 (25 nM) or co-treated with PK2 (25 nM) and 1 µM PC-7, a PKR1/PKR2 inhibitor, for 8 h (n = 4). (b) Western blot analysis probing for GDNF in U373 cells treated with PK2 (25 nM) for 10 h (n = 4). (c) qPCR analysis of GDNF mRNA from primary human astrocytes treated with PK2 for 4, 8, and 12 h (n = 2). (d) Immunocytochemical analysis probing for GDNF in primary human astrocytes treated with 25 nM PK2 for 10 h (n = 8; scale bar = 10 µm). (e) qPCR analysis of GDNF gene expression of U373 cells treated with IS20 (10 µM) for 1-6 h (n = 3). (f) GDNF mRNA levels measured by qPCR of U373 astrocytes transiently transfected with a PKR1 overexpression vector with increasing amounts of cDNA (0.5–5 µg) and treated with 10 µM IS20 for 4 h (n = 3–4). (g) qPCR analysis of PKR1 mRNA confirming CRISPR-Cas9 PKR1-KD. (h) qPCR analysis of GDNF mRNA in CRISPR-Cas9 PKR1-KD cells treated with IS20 (3–10 µM) for 8 h (n = 6). (i) GDNF promoter luminescence assay of U373 astrocytes transfected with 3.6 kb of GDNF promoter fragment and 24 h after transfection, treated with IS20 (5 µM or 10 µM) for 8 h. (j) GDNF promoter luminescence following 4 h, (k) 8h, and (l) 12h of 10 µM IS20 in U373 astrocytes transfected with GDNF promoter fragment. *p≤0.05, **p<0.01, ***p<0.001, ****<0.0001. Next, we sought to determine if IS20 pharmacological activation of PKR1 could modulate GDNF in U373 astrocytes. A time-course treatment of U373 cells with 10 µM IS20 revealed a 3-fold increase in GDNF mRNA after 1 h of IS20 incubation; this upregulation declined over the subsequent 5 h ( Fig. 1e ). To assess any receptor-specific effects of IS20 on GDNF production, we overexpressed PKR1 by transiently transfecting increasing amounts of PKR1 expression vector in U373 astrocytes. Ectopic expression of PKR1 (0.5–5 µg cDNA) dose-dependently stimulated the upregulation of GDNF mRNA in cells treated with 10 µM IS20 for 4 h ( Fig. 1f ). Importantly, when a CRISPR-Cas9 guide RNA against PKR1 was used to knock down PKR1 in U373 astrocytes ( Fig. 1g ), IS20 was no longer able to modulate GDNF expression ( Fig. 1h ), further demonstrating that IS20-induced GDNF expression is highly dependent on PKR1 receptor binding and activation. To validate our findings that IS20 could enhance GDNF expression and increase GDNF promoter activity, we utilized a GDNF promoter luciferase assay. U373 astrocytes were transfected with a 3.6 kb GDNF promoter fragment that encompasses GDNF promoter 1, TATA-box, the main transcriptional start site and exon 1 of the murine GDNF gene cloned into a pGL3 promoter vector that drives the expression of a luciferase reporter gene. 24 h after transfection, U373 cells were treated with 5 µM and 10 µM of IS20 for 8 h. We observed a dose-dependent increase in luciferase signal intensity with both doses significantly enhancing GDNF promoter activity ( Fig. 1i ). Next, we treated the transfected U373 cells with IS20 (10 µM) for 4, 8, and 12h. Notably, we observed a significant increase in relative luciferase signal intensity at all timepoints. Co-treatment of PKR1 antagonist, PKRA7, with IS20 attenuated the increase in the 4h and 8h groups while the reduction in GDNF promoter activity was not seen in the 12h group. This could be due to PKRA7 degradation after 12 h leaving it ineffective at blocking PKR1 activation. Together, these results suggest that IS20 can enhance GDNF promoter activity and gene expression in U373 astrocytes. GDNF is upregulated in primary mouse astrocytes and organotypic midbrain slice cultures treated with rPK2 and IS20 Next, we used cultured primary mouse astrocytes from prenatal mouse pups to validate the findings obtained from the human astrocyte cells. Treating primary mouse astrocytes with rPK2 (10–25 nM) for 8 h revealed a 3.4-fold upregulation of GDNF mRNA in the 25-nM group, which was abolished with PC-7 antagonism ( Fig. 2a ). Western blot and ELISA analysis confirmed a corresponding 1.5-fold increase in PK2 protein levels ( Fig. 2b–c ). Similarly, using primary mouse astrocytes stably expressing PK2 tagged with the green fluorescent protein (GFP) reporter ( Fig. 2d ), we found Lenti-PK2-GFP cells exhibited a 1.7-fold increase in GDNF protein levels compared to Lenti-GFP-infected control cells ( Fig. 2e ). Download figure Open in new tab Figure. 2 PK2 and IS20 increase GDNF in primary cultures of mouse astrocytes and organotypic slices. (a) qPCR analysis of GDNF mRNA in primary mouse astrocytes treated with PK2 (10–25 nM) or co-treated with PK2 and PC7 (1 µM) for 8 h (n = 6). (b) Western blot analysis probing for GDNF in primary mouse astrocytes treated with PK2 (25 nM) for 10 h (n = 3–5). (c) ELISA assay of GDNF protein from mouse primary astrocyte lysates treated with 25 nM PK2 for 10 h (n = 2). (d) Fluorescence microscopy showing stable expression of PK2-eGFP after lentiviral-mediated delivery of PK2 cDNA into primary mouse astrocytes. (e) Western blot analysis of GDNF protein level in primary mouse astrocytes transduced by lentiviral PK2-eGFP (n = 4). (f) qPCR assay of GDNF mRNA in primary mouse astrocytes treated with 10 µM IS20 for 1–2 h (n = 3). (g) GDNF ELISA of the supernatant of primary mouse astrocytes treated with 10 µM IS20 for 1–2 h (n = 3). (h) qPCR analysis of GDNF mRNA of primary mouse astrocytes treated with 0.5–30 µM IS20 for 3 h (n = 3). (i) GDNF ELISA of the supernatant of primary mouse astrocytes treated with 0.3–30 µM IS20 for 2 h (n = 3). (j) Western blot analysis probing for GDNF (left) in primary astrocyte lysates treated with 1 µM or 10 µM IS20 or 25 nM PK2 for 4 h or 8 h and densitometric analysis (right) of GDNF band normalized to β-actin (n = 2). (k) GDNF immunocytochemistry of primary human astrocytes treated with IS20 (10 µM) for 8 h (n = 4, scale bar = 10 µm). (l) qPCR analysis of GDNF gene expression in cultured midbrain organotypic slices treated with 0, 3, or 30 µM IS20 for 4 h (n = 3). (n) Western blot analysis of GDNF protein in cultured organotypic slices treated with 0 or 30 µM of IS20 for 4 h (n = 3). *p≤0.05, **p<0.01, ***p<0.001, ****p<0.0001. To evaluate the toxicity of IS20 on primary mouse astrocyte survival, we utilized the MTS assay. We found the LD50 toxicity at 24 h was between 348 µM and 592 µM IS20 (Supplementary Fig. 3a). To assess the GDNF response to IS20, we treated primary mouse astrocytes with 10 µM IS20 for 1–2 h. We found that IS20 induced rapid upregulation of GDNF mRNA, as measured by qPCR ( Fig. 2f ), and corresponded to a rise in GDNF protein in the media as measured by ELISA ( Fig. 2g ). A dose–response study was utilized to determine the optimal dose of IS20 in primary mouse astrocyte cells to elicit a GDNF response. Treating astrocyte cells with 0.5-to 30-µM IS20 for 2 h revealed that maximal GDNF expression occurred at 10 μM IS20 ( Fig. 2h–i ). Interestingly, mRNA of the predominant GDNF receptor in the DAergic system, GFRα1, was co-upregulated with GDNF in the 10-μM IS20 treatment group (Supplementary Fig. 3b), indicating that the increased gene expression and protein secretion of GDNF also lead to upregulated ligand–receptor interaction, possibly through a positive feedback loop, to promote GDNF signaling. GDNF protein levels were also increased in whole cell lysates treated with 1 and 10 µM of IS20 for 4-8 h ( Fig. 2j ). Moreover, ICC analysis confirmed the increase in GDNF following 8 h of 10 µM IS20, further demonstrating that GDNF is both upregulated and secreted by primary astrocyte cells following IS20 treatment. To evaluate the specificity of IS20 in stimulating GDNF expression, we used IS21, an analog of IS20 incapable of activating PKR1. Treating primary mouse astrocytes with IS21 for 3 h did not confer GDNF expression, while IS20 induced a 3-fold increase in GDNF mRNA (Supplementary Fig. 2b). Moreover, using Qiagen Neurotrophin and Receptor PCR array analysis, we found increased expression of 71 out of 84 genes in primary mouse glial cultures treated with IS20, including GDNF, CTNF, and BDNF (Supplementary Fig. 2c), further suggesting IS20 can promote neurotrophic support in mouse astrocytes. After establishing IS20’s effects on GDNF expression using astrocyte cultures, we validated those findings using an ex vivo approach employing organotypic midbrain slice cultures prepared from neonatal mouse pups. In line with our in vitro findings, treating organotypic midbrain slices with IS20 for 4 h increased GDNF mRNA expression ( Fig. 2l ) and protein levels ( Fig. 2m ). Collectively, these results demonstrate that GDNF is highly upregulated and secreted in both primary mouse astrocyte and midbrain slice cultures following PK2 and IS20 activation of PKR1 signaling. IS20 protects against MPP + -induced cell death and preserves mitochondrial energetics in DAergic neuronal cells Having discovered that IS20 is a potent inducer of GDNF in astrocytes, we further evaluated whether the IS20-induced upregulation and secretion of astrocytic GDNF could functionally protect DAergic neuronal cells under insult from the classic Parkinsonian toxicant MPP + . U373 astrocytes were incubated with or without IS20 for 2 h, followed by a change to fresh media and an additional incubation for 6 h. Both types of astrocyte-conditioned media (ACM) were collected and added to N27 DAergic neuronal cells that were then co-treated with or without MPP + (100– 300 µM) for 24 h ( Fig. 3a ). Adding IS20-treated ACM significantly increased cell viability in MPP + -treated N27 cells compared to MPP + -treated cells that had received only vehicle-treated ACM ( Fig. 3b ). Next, we investigated whether this protection was mediated through preservation of mitochondrial respiration. Seahorse (Agilent Technologies, Santa Clara, CA) analysis revealed that, in addition to slightly increasing the basal respiration of untreated N27 cells ( Fig. 3c ), IS20-treated ACM remarkably attenuated the MPP + -induced reduction in oxygen consumption rate, basal respiration and reserve capacity ( Fig. 3d ), suggesting that a post-mitochondrial mechanism at least partly contributes to the protection conferred by IS20-treated ACM. Lastly, we utilized mouse primary mesencephalic culture to validate the neuroprotective effects of IS20-treated ACM. We included the ACM collected from U373 astrocytes treated with IS21, an analog of IS20 that does not activate PKR1, to further determine the specificity of IS20. IS20-treated, but not IS21-treated, ACM promoted cell survival in MPP + -exposed mouse primary mesencephalic culture as determined by MTS assay ( Fig. 3e ). Altogether, these studies suggest that U373s treated with IS20 secrete factors that protect DAergic neuronal cells against MPP + -induced neurotoxicity. Download figure Open in new tab Figure 3. IS20 protects against MPP+-induced cell death and preserves mitochondrial energetics in DAergic neuronal cells. (a) Schematic of the IS20 treatment paradigm whereby U373 astrocytes were treated with 10 µM IS20 or vehicle control for 2 h, the media was changed and the U373 astrocyte cells were incubated for 6 h. Next, the astrocyte media was collected and added to N27 DAergic neuronal cells that were co-treated with or without MPP+ (100 µM or 300 µM). (b) MTS assay of N27 DAergic neurons after co-treatment with MPP+ and astrocyte-conditioned media (ACM) that had been incubated with or without IS20 for 24h (n = 6-7). (c–d) Seahorse Mito Stress Test of N27 cells treated with control ACM (grey line), IS20 ACM (yellow line), IS20 ACM added with MPP+ (blue line), and MPP+ alone (black line) (n = 12). (e) MTS assay of primary striatal culture isolated from the mouse and treated with the same control ACM, control ACM with MPP+, IS20 ACM + MPP+, IS21 ACM + MPP+, and the controls. *p≤0.05, **p<0.01, ***p<0.001, ****p<0.0001. IS20 administration induces GDNF expression and release in C57BL/6 mice Next, we determined the bioavailability of IS20 in the brain after intraperitoneal (i.p.) injection or IN administration and assessed its biological effects in the brain. First, wild-type C57BL/6 mice were i.p.-injected with DMSO or IS20 at 10 mg/kg body weight. Mice were sacrificed after 8 h to examine GDNF protein levels in the brain and serum by Western blot and ELISA, respectively. IS20 significantly increased GDNF protein levels in both nigral lysates ( Fig. 4a ) and serum ( Fig. 4b ). We next tested the IN route, which is known to concentrate small, lipophilic compounds more efficiently in the brain than i.p. injections. This is likely because the olfactory and trigeminal nerve pathways allow IN-administered compounds to bypass the blood–brain barrier 45 , thus increasing compound bioavailability in the CNS noninvasively while reducing exposure to peripheral tissues. Since we were expecting improved efficacy, we opted for a lower IN dose of 3 mg/kg IS20. Using liquid chromatography/mass-spectrometry (LC/MS), our pharmacokinetic study found that the concentration of IN-injected IS20 reached 4 ng/mg tissue in the whole brain at 30 min before gradually decreasing to 1.5 ng/mg tissue at 90 min and to <0.8 ng/mg tissue at 6 h ( Fig 4c ), indicating that IN-injected IS20 was able to cross the blood–brain barrier and accumulate in the brain. Interestingly, the brain level of IS20 remained relatively high even 24 h post-IS20 treatment. ELISA detected concurrent increases in GDNF protein in both brain lysates and serum ( Fig. 4d – e), with both responses peaking at 30 min post-IS20 treatment. Similar to i.p. injections of IS20, at 3 and 6 h post-IN administration of IS20, qPCR assays showed significant increases in GDNF mRNA expression in the SN and striatum ( Fig. 4f ). GDNF mediates pro-survival effects through its preferred receptor GFRα1, a glycosylphosphatidylinositol (GPI) protein, and its co-receptor RET, a receptor tyrosine kinase that transduces intracellular signals and whose decreased expression causes progressive degeneration of the nigrostriatal system 23 , 46 . Intriguingly, IS20 significantly increased the expression of GFRα1 and RET mRNAs in whole brain lysates ( Fig. 4g – h). Collectively, these data show that both minimally invasive i.p. delivery and non-invasive IN delivery of IS20 upregulate GDNF and GDNF receptors in the mouse brain. Download figure Open in new tab Figure 4. Intraperitoneal or intranasal administration of IS20 induces GDNF family neurotrophic factors in wild-type C57BL mice. (a) Western blot analysis probing for GDNF in substantia nigra (SN) lysates from mice intraperitoneally injected with 10 mg/kg IS20 8 h post-administration. (b) GDNF ELISA of blood serum 8 h after sacrifice of 10 mg/kg IS20-treated and vehicle-treated mice. (c) Mouse treatment paradigm (top) and liquid chromatography/mass spectrometry (LC/MS) analysis of IS20 in the brain of mice treated with IS20 (bottom). (d, e) ELISA assay of GDNF protein between 20 min to 24 h post intranasal IS20 (3 mg/kg) administration as measured in the (d) brain and (e) blood serum. (f) GDNF qPCR of the whole brain, SN, and striatum (STR) 3 h and 6 h after intranasal treatment of IS20 (3 mg/kg). (g, h) qPCR analysis of whole brain lysates collected 3 and 6 h post intranasal IS20 (3 mg/kg) administration, showing fold change in mRNA expression of (g) GFRa1 and (h) RET. *p≤0.05, **p<0.01, ***p<0.001. IS20 protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced DAergic cell death and preserves GDNF expression in C57BL/6 mice Since intracranial delivery of exogenous rGDNF has shown effective neuroprotection 47 , we hypothesized that the early IS20-mediated induction of striatal GDNF would protect DAergic neurons against neurodegeneration in a mouse model of PD. To test this hypothesis, we co-injected C57BL/6 mice with the classic Parkinsonian toxicant MPTP and IS20 i.p. once daily for 5 d, with IS20 being injected 1 h after MPTP. After 5 d of the MPTP–IS20 co-injections, we injected only IS20 for another 7 d ( Fig. 5a ). This intervention strategy of combining co-treatments with post-MPTP treatments allowed us to examine IS20’s effect on the early neurodegenerative process. As confirmed by qPCR, MPTP treatment alone mildly reduced GDNF gene expression, while IS20 co-treatment significantly protected GDNF gene expression in MPTP-treated mice ( Fig. 5b ). In concordance with our above-mentioned in vitro, ex vivo, and in vivo pharmacodynamics experiments, the largest fold-change in GDNF gene upregulation occurred in mice treated with IS20 alone ( Fig. 5b ). HPLC analysis of striatal homogenates showed that IS20 treatment modestly but significantly attenuated the MPTP-induced depletion of DA and its metabolites HVA and DOPAC ( Fig. 5c–e ). Finally, to further determine the extent to which IS20-driven GDNF upregulation protected the nigral–striatal system against MPTP-induced lesioning, TH immunohistochemistry (IHC) and stereological counts of TH-positive DAergic neurons were performed on caudate-putamen and SN cryosections obtained from mice in each group. As expected, while MPTP-alone significantly reduced the number of TH-positive DAergic neurons in the SN, IS20 significantly preserved the TH-positive neuron count in MPTP/IS20-cotreated mice ( Fig. 5f–g ). TH IHC also showed that IS20 treatment significantly preserved DAergic nerve fibers damaged by MPTP treatments ( Fig. 5g , representative sections from each group shown). These results suggest that IS20 treatment is protective against MPTP-induced neuronal and neurochemical deficits, possibly through enhanced GDNF signaling. Download figure Open in new tab Figure 5. IS20 protects against MPTP-induced DAergic cell death in C57BL/6 mice and restores GDNF expression. (a) Treatment paradigm for C57BL/6 mice intraperitoneally injected with MPTP and IS20 treatments for 5 d and 12 d, respectively. (b) qPCR analysis showing GDNF gene expression of mice after 12 d of treatments with vehicle control, MPTP alone, MPTP-IS20 co-treatment, or IS20 alone. (c–e) HPLC analysis of striatal lysates from mice treated with vehicle control, MPTP alone, MPTP-IS20 co-treatment, or IS20 alone showing (c) DA, (d) homovanillic acid (HVA), and (e) DOPAC. (f) Software-assisted stereological count of TH+ DAergic neurons in the SNpc of IS20 co-treated mice compared to the MPTP group. (g) DAB immuno-staining images of TH+ neurons in the SNpc under 10x magnification (scale bar = 200 µm). Student’s t-tests were used to evaluate MPTP vs. MPTP+IS20. *p≤0.05, **p<0.01. IS20 protects against behavioral deficits and DAergic neurodegeneration in the MitoPark mouse model of PD Because DAergic degeneration in the MPTP model stems from an acute neurotoxic insult and follows a rapid disease course, we next used the transgenic (Tg) MitoPark mouse model of PD, which more closely recapitulates PD’s chronically progressive neurodegenerative process. This genetic model of PD was rendered by cKO of mitochondrial transcription factor A (TFAM) driven by the Cre/LoxP system in DAergic neurons. The subsequent mitochondrial dysfunction results in PD-like symptoms, including a steady, progressive loss of DAergic neurons over several months, with the accompanying loss of motor capacity starting at around age 12–14 wk, as well as non-motor symptoms including olfactory deficits and depression. Littermates lacking the cKO of TFAM and DAT do not exhibit deficits and serve as ideal healthy controls. MitoPark mice age 13 wk were randomly chosen for IN treatment with vehicle or IS20 (3 mg/kg) for 4 wk ( Fig. 6a ). Download figure Open in new tab Figure 6. IS20 protects against behavioral deficits in MitoPark model. (a) Treatment paradigm for MitoPark mice treated with 3 mg/kg IS20 or vehicle via intranasal administration every other day for 28 d with biweekly behavioral monitoring using VersaMax. (b) Horizontal activity analysis of mice treated with IS20 (3 mg/kg) or vehicle after 14 and 28 d (n=2–3). (c) Representative tracing and heat plots of VersaMax analysis from 10-min activity monitoring of mice treated with IS20 (3 mg/kg) or vehicle after 14 and 28 d. *p≤0.05. Using this model allowed us to test if a more chronic IN treatment regimen attenuates the onset and progression of behavioral and motor deficits in a more gradually progressive model. Starting at 15 wk (14 d into treatment), control MitoPark mice exhibited a drastic reduction in locomotor activity ( Fig. 6b–c ). However, MitoPark mice that received IN IS20 exhibited slightly more horizontal activity compared to vehicle controls, and by 28 d post-treatment, MitoPark mice that received IS20 performed significantly better ( Fig. 6b–c ). The MitoPark mice were then sacrificed for tissues after treatment at age 17 wk. The SN of IS20-treated MitoPark mice showed significantly increased GNDF gene expression, as well as GDNF protein expression, as measured by qPCR and Western blot, respectively, when compared to vehicle-treated mice ( Fig. 7a–b ). TH protein levels in the MitoPark SN were also significantly protected by IS20 treatment, whereas PKR1 levels were unaffected ( Fig. 7b–c ). Since astrocyte-derived GDNF can reduce microglia-induced neuroinflammation 48 , 49 , we also assessed IBA1 protein levels and found a decrease in expression in the SN ( Fig. 7b–c ), potentially indicating a reduction in microglial activation in IS20-treated mice. To confirm the functional effects of IS20-induced GDNF upregulation on DAergic neurodegeneration in MitoPark mice, brain sections from 16-wk MitoPark mice were immunostained for TH to detect DAergic neurons. DAB immunostaining and stereological counts of TH-positive DAergic neurons revealed significantly higher neuronal counts in the SN when comparing IS20-to vehicle-treated MitoPark mice ( Fig. 7d ). Taken together, these experiments show that IS20 IN delivery could significantly protect against DAergic neuronal cell loss and behavioral deficits in the Tg MitoPark mouse model of PD. Download figure Open in new tab Figure 7. IS20 protects against DAergic neurodegeneration in MitoPark model. (a) qPCR of GDNF and of SN lysates from 13-wk-old MitoPark mice treated intranasally with IS20 (3 mg/kg) or vehicle control every other day for 28 d. (b) Western blot analysis probing for GDNF, TH, IBA1, and PKR1 in SN brain lysates from IS20-or vehicle-treated MitoPark mice; β-actin was used as loading control (n = 6). (c) Densitometric analysis of Western blots (shown in b) normalized to β-actin. (d) Tyrosine hydroxylase (TH) immunostaining of SN sections imaged at 2X (top) and 10X (bottom) magnifications with computer-assisted stereological counting of TH+ cells in the SNpc (right). *p≤0.05, **p<0.01, ***p<0.001 when compared to vehicle control group. IS20 activates AKT and p44/42 MAPK signaling pathways and induces pro-survival Bcl2 gene expression Knowing that AKT phosphorylation might induce GDNF upregulation, we explored IS20’s effects on cellular signal transduction in primary mouse astrocytes. IS20 rapidly induced both AKT and p44/42 phosphorylation ( Fig. 8a ) and promoted the expression of Bcl2 mRNA ( Fig. 8b ). Furthermore, in C57BL/6 mice 8 h after IP injection, IS20 induced AKT phosphorylation in the striatum ( Fig. 8c ). These experiments suggest mechanistically that IS20 promotes signaling transduction known to induce GDNF upregulation as well as other neuroprotective factors. Download figure Open in new tab Figure 8. IS20 activates p44/42 and AKT pathways and induces pro-survival NRF2 and BCL2 gene expression. (a) Western blot analysis probing for phosphorylated AKT and phosphorylated p44/42 in primary mouse astrocytes treated with IS20 for 0.5–6 h. (b) qPCR of Bcl2 gene expression in primary mouse astrocytes treated with IS20. (c) Western blot analysis probing for phospho-AKT and AKT in striatum lysates from C57BL/6 mice at 8 h after IP injection of IS20. *p≤0.05, **p<0.01. Discussion In this study, we demonstrated the potential for a small-molecule PKR1 receptor agonist to induce GDNF expression and to achieve neuroprotective effects in two rodent models of PD. In particular, we show that activation of PKR1 signaling through IS20 treatment quickly causes GDNF secretion and gene expression in primary astrocyte and organotypic slice cultures. In our experiments with C57BL/6 mice, IN-administered IS20 successfully crossed the blood–brain barrier, reached maximum concentration in around 30 min, and induced GDNF serum release and gene expression in the nigrostriatal system. Using a subacute MPTP mouse model of PD, we show that IS20 protects DAergic neurons against MPTP-induced neurodegeneration and helps to preserve striatal DA levels. Furthermore, we show in Tg MitoPark mice that a 4-wk course of IN-administered IS20 induced GDNF and TH levels in the SN, suppressed microglial activation, protected against DAergic neurodegeneration, and that these protective effects translate to functional improvements in locomotor activity for MitoPark mice. These findings suggest that pharmacological modulation of GDNF signaling is achievable with minimal side effects using IN administration of the PKR1 receptor agonist IS20. PK2 activates PKR1 at an EC 50 of 4.3 ± 1.3 nM and PKR2 at an EC 50 of 7.3 ± 2.8 nM 50 . The apparently similar receptor affinities 51 suggest that downstream effects induced by PK2 are determined by the expression levels of PKR1 and PKR2 on the cell surface 52 . We previously showed that PK2 signaling through PKR1 and PKR2 in neurons rapidly increases intracellular Ca 2+ and induces phosphorylation of AKT and p44/42, leading to increases in BCL2 protein levels and mitochondrial biogenesis. We also developed an efficient AAV2/5-mediated gene delivery system, which produces robust, stable expression of PK2 in the striatum and protects SN neurons from MPTP-induced neurodegeneration. In astrocytes, activation of prokineticin signaling is likely modulated by the predominant expression of PKR1 36 . Our current study shows that the PKR1 receptor agonist IS20 rapidly increases intracellular Ca 2+ and induces phosphorylation of AKT and p44/42 in astrocytes. AKT activation is a known pathway that results in secretion and upregulation of endogenous GDNF. Since both prokineticin and GDNF signaling depend on AKT and ERK signaling in DAergic neurons to mediate protective effects 35 , 46 , it is therefore possible that crosstalk between prokineticin and GDNF signaling mediates IS20’s pro-survival effects. Indeed, Tanabe et al. (2012) 53 report that blocking AKT and p44/42 using API and PD98059, respectively, in PK2-treated neurons nullifies PK2’s protective effects, while blocking p44/42 using PD98059 in rat glioma cells prevents GDNF release. Their findings further suggest that prokineticin and GDNF signaling converge at AKT and p44/42 in DAergic neurons to promote neuronal survival. Yamagata et al. (2002) 54 revealed that GDNF is upregulated by ischemia and that astrocyte-derived GDNF protects neuronal cultures from cell death in cases of mitochondrial damage (see also C.-H. Lin et al., 2006). PK2 is also upregulated by hypoxia in primary cortical culture models of ischemia 55 ; induces proliferation, survival and migration of capillary endothelial cells under hypoxic stress 56 ; and supports mitochondrial biogenesis under MPP + -induced oxidative stress 35 . These previous findings support our hypothesis that PK2 is co-regulated in the brain with GDNF. Others also report significant crosstalk between PK2 and GDNF signaling in the development of the ENS 32 . Both PK2 and GDNF are also strong chemoattractants for projecting axons during neurogenesis and neuritogenesis. Additionally, PKR1 cKO leads to heart and kidney disorders due to deficits in angiogenesis, cell survival signaling, and mitochondrial and progenitor cell functions in these organs 57 . The involvement of both GDNF and PK2 signaling in PNS and CNS functions suggests crosstalk between their pathways. Previous GDNF clinical trials concluded that although the potential for GDNF-based therapy remains high, two challenges need to be overcome: proper selection of animal-based PD models during preclinical testing and unwanted compensatory reactions or side effects associated with GDNF delivery 58 . MPTP has been the most frequently used Parkinsonian toxicant applied in the generation of animal models of PD 59 , 60 , ever since the accidental discovery decades ago of its ability to recapitulate the disease in humans 61 , 62 . Although subacute MPTP treatment (3–5 daily injections) in rodents produces PD-related neuropathology, this outcome does not progress gradually nor does it reliably reproduce the suite of behavioral deficits characterizing the human disease 63 – 65 . MPTP injections leave striatal DAergic neuron projections intact, which are then capable of retrogradely transporting GDNF to their somata in the SN. Yet, recent clinical data observed in post-mortem brains of PD patients 5 y post-diagnosis revealed that TH-positive DAergic innervations from the caudate–putamen completely disappeared, questioning the effectiveness of delivering GDNF only in the striatum of advanced PD patients, as employed by clinical trials conducted by Amgen 66 – 68 and others 69 , 70 . Thus, the translational value of past studies relying only on acute neurodegeneration models that suggest targeting the striatum is both necessary and sufficient is confounded by their intact axonal transport machinery 71 . In contrast, MitoPark mice used in the current study lost striatal projections at age 17 wk, recapitulating conditions seen in advanced PD patients, thereby allowing critical assessments of preclinical functional endpoints. This current understanding sheds light on earlier studies reporting that GDNF administration by intranigral injection could mitigate neuronal cell loss, but did not protect against loss of terminal projections 56 , 72 . And by extension, intrastriatal injection of AAV-GDNF could protect against degeneration of both neuronal cell bodies and projections if administered before neuronal cell loss 73 . This is presumably because GDNF must be retrogradely transported to the neurosomata for RET signaling to mediate pro-survival effects 23 . Therefore, high GDNF expression in the striatum is less effective if GDNF’s retrograde transport is disrupted due to significantly degenerated DAergic neuronal projections. In this case, increasing GDNF levels in both the striatum and SN might prove to be more effective in promoting GDNF-induced pro-survival effects 74 . Indeed, success has been achieved using AAV2-mediated neurotrophic factor gene transfer in both the striatum and nigra 73 , 75 . In our current study, IS20-induced GDNF expression levels were upregulated in both the striatum and SN of MitoPark mice, allowing astrocyte-secreted GDNF to protect both DAergic projections in the striatum as well as neurosomata in the SN. Significantly more striatal projections are preserved in MitoPark mice treated with IS20. Together, these data indicate that performing preclinical studies in animal models that recapitulate the gradual progression of PD pathology could improve the transition from translational research to clinical trials for PD and reduce the attrition of candidate therapies. Most current GDNF gene delivery trials use AAV2, which has low immunogenicity and has proven safe for use in humans, but transduces predominantly neurons 76 , 77 . However, in the injured brain, GDNF is mainly produced by astrocytes, not by neurons 78 , 79 , as when astrocytes secrete GDNF during ischemia as a compensatory response 80 . Astrocytes also provide the major source of GDNF in the SN of PD patients’ midbrains 28 , 29 , where astrocytes and microglia outnumber neurons 10 to 1 49 , 81 . Early studies isolated nigral GDNF from Type 1 astrocytes and found that astrocyte-derived GDNF enhanced DAergic neuron survival when nigral astrocytes were co-cultured with DAergic neurons as support cell monolayers 11 . Astrocytes are also known to secrete other neurotrophic factors, such as CDNF, BDNF, and NGF. Moreover, in the gut, enteric glia cells are the major source of GDNF 82 , 83 . Therefore, neuroprotective efforts targeting only neurons for long-term neuronal viability are unlikely to succeed if supportive astrocytes do not provide proper neurotrophic and metabolic enrichments 84 . Several studies have found that intrastriatal, viral-mediated GDNF gene delivery causes aberrant neurite growth towards the site of application 23 , 39 , suggesting that mimicking the endogenous mode of expression is important to achieve functional effects. Drinkut et al. (2012) 28 focused on viral delivery of GDNF into astrocytes, achieving localized, yet satisfactory neuroprotection. Building upon these studies, we found here that targeted GDNF upregulation in astrocytes could be achieved pharmacologically using a non-invasive route of administration, suggesting that additional research efforts should be redirected back to astrocytes. The neuroprotective effects afforded by IS20 could also be mediated through the reduction of microglial IBA1 expression in the SN, which has a high density of microglia 85 , whose activation is affected by PD pathology 86 . But here, too, the role of astrocytic GDNF appears to extend to neuroinflammation associated with neurodegeneration as it can potently inhibit excessive production of reactive oxygen species from microglia in zymosan A-stimulated midbrain microglia cultures 49 and LPS-stimulated primary midbrain neuron–glia cultures 87 . Thus, astrocyte-derived GDNF could mediate protective effects by its anti-inflammatory effects on activated microglia 86 . In line with previous studies, we show that IS20 treatment reduced IBA1 expression in the SN of 17-wk-old MitoPark mice. However, constitutive GDNF expression from viral transgene expression can downregulate TH expression 24 , possibly due to feedback loops between GDNF and DA reuptake 88 , 89 . To counteract such unwanted compensatory effects, a discontinuous GDNF delivery paradigm could allow GDNF to return to basal levels 28 , 58 . Our pharmacodynamic studies in healthy control mice found that IS20-induced GDNF expression in the striatum and SN, as well as the whole brain, returned to basal levels 6 h post-treatment, thereby avoiding constitutive GDNF expression. Consequently, we saw increases in TH in MitoPark mice treated with IS20. Yet another possible cause of an unwanted compensatory effect, driven by viral transgenes in other studies 26 , results from GDNF expression that is at least one or two orders of magnitude higher, In our study, we have seen 2-fold and 3-fold increases in GDNF expression induced by IS20 in the striatum and SN, respectively, and a 0.5-fold increase in GDNF expression in whole brain relative to control levels. Furthermore, we did not see overt signs of toxicity or a significant reduction in body weight with the administered doses, routes, and study duration. This is in agreement with a previously published study, where subchronic i.p. administrations of PK2 protein, which activates PKR1 and PKR2 in a relatively non-selective fashion 90 , were administered systemically in C57BL/6 mice without causing serious toxicity 91 . In summary, IS20 administration could pharmacologically upregulate GDNF levels in the nigrostriatal pathway, preserving DAergic neurons and functional innervations in neuroprotective paradigms applied in PD animal models. Importantly, the relative safety profile of IS20 makes it a promising pharmacological candidate for PD therapy. Materials and Methods Calcium Assays Intracellular calcium mobilization was assessed using the Fluo-4 NW Calcium Assay Kit (F36206, Thermo Fisher, Waltham, MA) according to manufacturer’s instructions. Briefly, CHO cells were grown on 96-well plates the previous day. FLIPR (Molecular Devices, San Jose, CA) was used to inject dissolved IS20 into the cell plate wells, which were read on a FlexStation multi-mode microplate reader (Molecular Devices). Animal handling All animal procedures were approved by Iowa State University’s Institutional Animal Care and Use Committee (IACUC). All mice were housed under a 12-h light cycle in a climate-controlled mouse facility (22±1 °C) with food and water available ad libitum . Male C57BL/6 mice were pre-screened during behavior assessments for normal baseline performance before being randomly assigned to experimental groups. None of the animals manifested wounds or any abnormal behaviors that would have warranted their removal from the study. Investigators involved with data collection and analysis were not blinded to group allocation. MPTP injections Over a 5-day period, one i.p. injection of 18 mg/kg MPTP was administered every day to each mouse in the MPTP group, and an equal volume of saline (vehicle) was similarly injected into each mouse in the control group. Behavior monitoring All groups were monitored for voluntary, exploratory locomotor activity using the automated VersaMax system and (analyzer, model VMAUSB, AccuScan, Columbus, OH) connected to VersaMax motion-detection hardware (monitor, model RXYZCM-16). The 40×40-cm square monitor was partitioned into four, 20×20-cm square open-field arenas. The VersaMax system is capable of tracking two animals simultaneously when using two diagonally opposed arenas that did not share a wall of infra-red (IR) beams. Each animal was allowed to acclimate to its arena for 2 min, after which its spontaneous locomotor activities (horizontal activity, vertical activity, and speed), monitored as IR beam-breaks, were recorded for 10 min. Fusion software was used to generate heat maps (Omnitech Electronics, Columbus, OH). MitoPark mouse model of PD MitoPark mice were a kind gift from Dr Nils-Göran Larsson of the Karolinska Institute, Stockholm, Sweden, from his laboratory at the Max Planck Institute for Biology of Ageing. The Tg MitoPark mouse model is created by TFAM inactivation specifically in DA neurons by cKO through control of the DA transporter promoter. The mice used were from our MitoPark breeding colony at Iowa State University. After behavioral experiments were performed at the predetermined ages, mice were euthanized at the predetermined endpoints via CO 2 asphyxiation, as outlined in approved IACUC protocols, and samples processed for either RT-qPCR or Western blotting. Western blot Dissected brain regions were collected in Eppendorf tubes and flash frozen. To isolate total protein, RIPA buffer containing sodium orthovanadate and Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher 78440) was added to each tube and homogenized using a tissue homogenizer. Dissolved total lysate was centrifuged at 12,000 x g for 60 min to remove cellular debris. Normalized protein samples were loaded into each well of a 12-well plate and were separated by sodium dodecyl sulfate (SDS) gel electrophoresis (100 V, 90 min), using Any kD Mini-PROTEAN resolving gel (4569035, Bio-Rad, Hercules, CA). Proteins on SDS gels were then transferred to a nitrocellulose membrane (26 V, overnight) and blocked for 1 h using fluorescent Western blocking buffer (Rockland Immunochemicals, Pottstown, PA). Primary antibodies diluted in blocking buffer with 0.05% Tween 20 were then added to the membranes and incubated overnight at 4 °C. Next day, primary antibodies were removed, and blots were washed (7 times, 5 min each) in PBS wash buffer containing 0.05% Tween 20 (PBST). Secondary antibody (infrared dye-tagged) was added for 1 h. Blots were then triple-washed in PBST and once in PBS. The protein β-actin was used as a loading control. Membranes were scanned using the Odyssey IR imaging system (LI-COR, Lincoln, NE) and digital images were captured via a LI-COR Odyssey imager. Densitometric analysis was done using ImageJ software. IHC Cells were fixed in 4% paraformaldehyde, blocked with BSA and Triton-X to permeabilize the cell membranes. Primary antibodies were diluted as follows: rabbit anti-TH (1:1600), rabbit anti-PK2 (1:500), goat anti-GDNF (1:500), goat anti-IBA1 (1:1000), and rabbit anti-GFAP (1:1000). Alexa Fluor fluorescent secondary antibodies (Thermo Fisher) were used against primary antibodies for fluorescent imaging. For DAB staining, HRP-conjugated secondary antibodies were used against primary antibodies, and VECTASTAIN Elite ABC HRP Kit (Vector Labs, Newark, CA) was used for conjugating HRP to secondary antibodies. DAB was used for colorimetric detection. SYBR Green RT-qPCR To obtain total RNA, tissue lysis buffer from the Absolutely RNA Miniprep kit (Agilent Technologies) was added to dissected, region-specific brain tissue samples together with beta-mercaptoethanol as a reducing agent to preserve RNA. A tissue homogenizer was used to dissolve tissue into the lysis buffer, and lysates were processed according to kit manufacturer’s instructions. Total RNA isolated from each sample was quantified using NanoDrop (Thermo Scientific) to determine RNA concentration and purity. First, strand cDNA synthesis was performed using the Affinity Script qPCR cDNA synthesis system (Agilent Technologies) with 1 µg of total RNA in the reaction mixture. Real-time PCR was performed with the RT 2 SYBR Green master mix (Qiagen, Germantown, MD) using diluted cDNA and QuantiTect (Qiagen) mouse primer sets. The 18S rRNA gene (mouse) was used as the housekeeping gene to normalize each sample. The amount of each template was optimized empirically to maximize efficiency without inhibiting the PCR reaction. After the last cycle of the qPCR run, dissociation curves were run to ensure a single amplicon peak was obtained, indicating primer specificity. The results are reported as fold change in gene expression derived with the ΔΔC t method, using the threshold cycle (C t ) value for the housekeeping gene and for the respective gene of interest in each sample. Control animals serve as the baseline for fold change. Lentivirus production The PK2 lentivirus expression vector (OriGene, Rockville, MD) was mixed with the MISSION Lentiviral Packaging Mix (SHP001, Sigma, St. Louis, MO) according to manufacturer’s protocols, and co-transfected into 293FT cells to package the virus. After 24 h post-transfection, the supernatant, which contains the virus, was collected. The second harvesting of virus was done 48 h post-transfection. The Lenti-X™ p24 Rapid Titer Kit (632200, Clontech, Mountain View, CA) was used to titer the lentivirus. HPLC analysis of striatal DA levels HPLC samples were processed as previously described 35 (Gordon et al., 2016). Briefly, mice were euthanized, striata were collected and neurotransmitters were extracted in 0.2 M perchloric acid solution containing 0.05% Na 2 EDTA, 0.1% Na 2 S 2 O 5 and isoproterenol (internal standard). DA and metabolites were separated isocratically by a reversed-phase column with a flow rate of 0.6 ml min −1 using a Dionex Ultimate 3000 HPLC system (pump model ISO-3100SD, Thermo Scientific) equipped with a refrigerated automatic sampler (model WPS-3000TSL). The CoulArray (model 5600A) electrochemical detection system was coupled with an analytical cell (microdialysis cell model 5014B) and a guard cell (model 5020). Data acquisition and analysis were performed using Chromeleon 7 and ESA CoulArray 3.10 HPLC Software. Data analysis All in vitro data were determined from at least two biologically independent experiments, each done with a minimum of three biological replicates. Data analysis used Prism 10.5.0 software (GraphPad Software, San Diego, CA) to perform one-way ANOVA and then Bonferroni’s post-tests or two-way ANOVA to compare all treatment groups. Student’s t-test (two-tailed) was used when two groups were being compared. Differences with p≤0.05 were considered statistically significant. Conflict of Interest All authors declare no actual or potential competing financial interests. A.G.K. has an equity interest in Probiome Therapeutics. The terms of this arrangement have been reviewed and approved by the University of Georgia in accordance with their conflict-of-interest policies. Acknowledgements This work was supported by National Institutes of Health grant R01 ES034196. 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