Validity of the MPTP-Treated Mouse as a Model for Parkinson’s Disease

Parkinson’s disease (PD) is characterized by dopaminergic (DA) neuron death in the substantia nigra (SN) and subsequent striatal adaptations. Mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine (MPTP) are widely used as a model for PD. To assess the validity of the MPTP mouse model for PD pathogenesis, we here identify the biological processes that are dysregulated in both human PD and MPTP-treated mice. Gene enrichment analysis of published differentially expressed messenger RNAs (mRNAs) in the SN of PD patients and MPTP-treated mice revealed an enrichment of gene categories related to motor dysfunction and neurodegeneration. In the PD striatum, a similar enrichment was found, whereas in the striatum of MPTP mice, acute processes linked to epilepsy were selectively enriched shortly following MPTP treatment. More importantly, we integrated the proteins encoded by the differentially expressed mRNAs into molecular landscapes showing PD pathogenesis-implicated processes only in the SN, including vesicular trafficking, exocytosis, mitochondrial apoptosis, and DA neuron-specific transcription, but not in the striatum. We conclude that the current use of the MPTP mouse as a model for studying the molecular processes in PD pathogenesis is more valid for SN than striatal mechanisms in PD. This novel insight has important practical implications for future studies using this model to investigate PD pathogenesis and evaluate the efficacy of new treatments. Electronic supplementary material The online version of this article (doi:10.1007/s12035-015-9103-8) contains supplementary material, which is available to authorized users.


FIGURE DESCRIPTIONS
Parkinson's disease (PD) as well as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment result in the degeneration of dopaminergic neurons in the substantia nigra (SN), leading to a decrease of dopamine (DA) release to the striatum. Below, the molecular landscapes of biological processes shared between PD and MPTP treatment in the substantia nigra (SN) (Figures 1a and 1b) and striatum (Figure 2) are described in full detail, and the current knowledge about the functions and interactions of all landscape proteins is presented. In these descriptions, proteins that appear in bold are dysregulated in both human PD and the MPTP mouse model. Underlined proteins are associated with PD through either expression or genetic data from PD patients, and familial PD proteins are double underlined. Figure 1A.Molecular landscape of interacting proteins, encoded by the mRNAs that are differentially expressed in the SN of both human PD patients and MPTP-treated mice, located primarily in the (pre) synapse and axon of the DA neuron.

Detailed description of the biological processes depicted in
DA synthesis TH catalyzes the rate-limiting step in DA synthesis, i.e. the conversion of the amino acid L-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA) [1,2]. TH expression is increased by the receptor tyrosineprotein kinase RET[3] and SLC6A3[4]. TH is activated by the adaptor protein YWHAZ[5,6] and the cyclin-dependent kinase CDK5[7], and DA itself inhibits TH activity in a negative feedback loop [8,9]. YWHAZ also binds to the kinases MARK2[10], involved in microtubule regulation [11], and PDXK[12], required for the synthesis of pyridoxal-5-phosphate (PLP) from vitamin B6 [11]. PLP, in turn, is an essential cofactor for the conversion of L-DOPA into DA by dopa decarboxylase [13].
(DA) release Soluble NSF Attachment Protein Receptor (SNARE) proteins form a complex that is required for synaptic vesicle docking and subsequently the release of their contents (e.g., DA) into the synaptic cleft [11]. SNAP25 and VAMP2, core components of the SNARE complex, physically interact with each other [14] and form a complex together with STXBP1[15], a protein that is also involved in synaptic vesicle fusion and docking [11]. STXBP1 binds to CDK5[16], a protein involved in cytoskeleton regulation, synapse plasticity, exocytosis and endocytosis (see more on CDK5 below). The vesicle fusion ATPase NSF increases the dissociation of the SNARE complex (dissociation of SNAP25 and VAMP2) and thereby enables the vesicle to fuse with the plasma membrane [17]. NSF binds to YWHAZ[18] and AKT1 [19], and inhibits CASP3 activity [20].
The DA -acetylcholine (ACh) balance may be involved in PD pathology [21] and ACh regulates DA release [22]. The extracellular protein ACHE hydrolyzes ACh that is released into the synaptic cleft [11]. Although ACHE does not directly interact with other landscape proteins, its function may well be linked to PD since its activity is reduced in the cerebral cortex and the medial occipital cortex of PD patients [23]. Moreover Microtubule-dependent trafficking In addition to RAC1, FYN is a non-receptor tyrosine-protein kinase that is involved in cell growth and survival, cell adhesion, cytoskeletal remodeling and axon guidance [11]. FYN is activated by L1CAM [96] and is itself an activator of RAC1 (see above), TNK2 [97] and CASP3 [98]. FYN is cleaved by CASP3 [99,100]and binds to TNK2 [97], GABRG2 [101] and MAPT [102]. Like FYN, TNK2 is a nonreceptor tyrosine-protein kinase and is involved in cell survival, proliferation and endocytosis [11], whereas GABRG2 is a subunit of the GABA receptor and regulates neuronal inhibition [11]. Moreover, FYN phosphorylates MAPT [103,104], which is a susceptibility gene for idiopathic PD [105][106][107][108][109] that promotes assembly and stability of microtubules [11]. Microtubule-dependent trafficking is affected in PD and, among others, affects axonal transport of autophagosomes that contain damaged mitochondria and aggregated proteins, which can lead to SNCA accumulation and synaptic dysfunction [110,111] [117]. Furthermore, MARK2 phosphorylates both MAPT and MAP4 which causes microtubule detachment and disassembly [11]. CHP1 binds to microtubules and mediates the binding of the endoplasmic reticulum (ER) and the Golgi apparatus with microtubules (not shown) [11]. Other proteins in the landscape that bind microtubules are MAPRE2 (not shown) [118], NDRG1 (not shown) [119], KLC1 (not shown)[11] and MAP4 (not shown) [120]. KLC1 is a kinesin that regulates microtubule-associated transport of organelles[11] and MAP4 promotes the assembly of microtubules [121]. In addition, MAPK8 is known to increase microtubular assembly [122,123].  [139]. TSPAN7 is a surface glycoprotein that may have a role in neurite outgrowth [143]. MAP4 and MARK2 regulate microtubular dynamics (see above). SLC4A3 is an anion exchanger that exchanges HCO3for Cland thereby regulates the intracellular pH [11]. Another protein in the PD landscape that regulates neuronal pH by transporting HCO3-into the cell is SLC4A8[11]. CRMP1 regulates remodeling of the cytoskeleton [11]. PFKM binds to YWHAZ[18] and catalyzes the conversion of D-fructose 1,6-phosphate to D-fructose 1,6biphosphate [11]. Binding of D-fructose 1,6-bisphosphate to soluble Fe2+ prevents its conversion to the insoluble Fe3+, an oxidation step that produces oxygen radicals. The availability of D-fructose 1,6biphosphate may therefore affect iron content and oxygen radical levels [144] in the SN of PD patients. Lastly, RAB14 and SNAP25 are involved in intracellular trafficking (see above). Furthermore, VDCC function and thus calcium influx is inhibited by CDK5 [145,146]. LPAR1 increases calcium mobilization in the cytosol [147], whereas RGS4 and RGS7 both decrease mobilization of calcium [148,149]. In addition, RAB4A and RAB11A (not shown) increase the intracellular calcium concentration [150]. Calcium in turn activates MAPK8 [ [166], inhibits TH [158,167] and MAPK8 [168], and is inhibited itself by FYN [162]. Interestingly, SNARE (SNAP25 and VAMP2) dysfunction results in mislocalization and accumulation of SNCA and could be an important pathomechanism of PD [169], which emphasizes the importance of the normal functioning of the SNARE complex. Furthermore, binding of PARK7 to VAMP2 [170] and of LRRK2 to NSF [171] shows that other familial PD proteins also have a direct impact on SNARE complex function. LRRK2 also binds to MAP2K4 [172], GNAI2 [173] and YWHAZ [174], and activates AKT1 [175] [182] and MARK2 binds to [183], and activates, PINK1 [183]. Figure 1B. Molecular landscape of interacting proteins, encoded by the mRNAs that are differentially expressed in the SN of both human PD patients and MPTP-treated mice, located primarily in the cell body and axon of the DA neuron.
Alternative pre-mRNA splicing. The polymerase PAPOLA creates the 3'-poly(A) tail of mRNAs[11], is required for endoribonucleolytic cleavage at poly(A) sites[11] and binds to HDAC1 (see above). YTHDF2 has also a role in mRNA stability and splicing, by binding to N6-methyladenosine [11]. Of note, multiple other proteins involved in mRNA splicing are dysregulated in both human PD and the MPTP mouse model. MAGOH1 and CASC3 are core components of the exon junction complex that is deposited at splice junctions on mRNAs, regulating mRNA splicing, nuclear export, cellular localization and translation efficiency [11]. MAGOH1 binds to CASC3 [251], ZC3H11A [251], SRSF7 [251], RBM39 [251], and SRPK2 (not shown) [252]. RBM39 also binds to SRSF7 [253] as well as YWHAZ [182] and SRPK2 [254]. SRPK2 is required for spliceosome complex formation [255] and, together with MAGOH1 and RBM39, binds to SRSF7 [252] and MAPT [256], and increases the phosphorylation of RBM39 (not shown) [252], SRSF7 (not shown) [252] and MAPT (not shown) [256]. Phosphorylation of SRPK2 at Thr-492 by AKT1 promotes its nuclear translocation and enhances its activity [11]. Like CASC3, MAGOH1 and SRPK2, RBM39 and SRSF7 are involved in pre-mRNA splicing [257,258]. For instance, SRSF7 is involved in mRNA export out of the nucleus [259] and is known to prevent splicing of exon 10 of MAPT (not shown) [260]. CLK4 phosphorylates proteins of the spliceosome complex[11] and regulates the alternative splicing of MAPT [261]. MAPT itself increases the expression of MAPK8 [262]. Other proteins that also affect alternative splicing and are involved in nucleosome/ histone regulation are HNRNPH3, CRMP1, H2AFJ and ANP32B. HNRNPH3 associates with pre-mRNA in the nucleus [11], and binds to PARK2 [263] and CRMP1 [264]. H2AFJ is a H2A histone variant and core component of the nucleosome[11] and ANP32B stimulates core histones to assemble into a nucleosome [11]. Nucleosomes define the exon-intron border and since pre-mRNA splicing occurs cotranscriptionally, nucleosome organization, transcription elongation rate or epigenetic marks can affect pre-mRNA splicing [265,266]. Moreover, histone deacetylation by HDAC1 affects pre-mRNA splicing, resulting in local repression of transcription [267,268,265]. HDAC1 is up-regulated in the SN of human PD patients and interacts with multiple proteins in the landscape (see also above). Taken together, the central position of HDAC1 and the occurrence of multiple proteins involved in histone regulation and pre-mRNA splicing in the SN landscape suggest that dysregulation of nucleosome organization and the splicing machinery are important factors in the biological processes that overlap between PD and the MPTP mouse model.
(Vesicle) trafficking and exocytosis In Figure 1a, the involvement of the SNARE complex in (DA) exocytosis is shown, however, the SNARE complex also regulates intracellular transport, as is apparent from the binding of SNAP25 to both NAPB [269] and KLC1 [270]. NAPB is required for vesicular transport between the ER and the Golgi apparatus [11], and KLC1 is a microtubule-associated protein that regulates the transport of organelles such as mitochondria. Like the SNARE complex, the familial PD protein SNCA may be involved in DA release and transport [11], but also in ER-to-Golgi vesicle trafficking [271,272]. SNCA modulates vesicle trafficking by binding to RABAC1 (not shown) [273], a protein that regulates the interaction between Rab GTPases and the SNARE complex [274]. Overexpression of SNCA disrupts vesicle trafficking and increases accumulation of vesicles in the cytoplasm [273]. Four Rab GTPases (RAB4A, RAB6A, RAB11A and RAB14) are overlapping between PD and the MPTP mouse. These proteins are involved in vesicular trafficking between compartments of the cell. RAB4A regulates localization of VAMP2 to early endosomes and vesicles [275] and the membrane-bound form of RAB4A binds to NDRG1 [276], a protein that is required for vesicular recycling [11]. NDRG1 binds to actin filaments by binding to ACTG1[91] as well as to ACOT7 [277] and PPP2R2A[91], and activates CASP3 [278]. The RAB proteins RAB6A, RAB11A and RAB14 are located in the Golgi complex and regulate protein trafficking to other organelles and the plasma membrane of the cell. Dysfunctioning of these proteins results in defective protein trafficking and membrane fusion, which can result in protein aggregation. RAB6A is located at the Golgi [279] and regulates vesicular transport from early and recycling endosomes to the Golgi (not shown) [280] but also transport from the Golgi to the ER [281]. Furthermore, RAB6A affects release of the SNARE (SNAP25 and VAMP2) complex, which itself is involved in membrane fusion (see also Figure 1a) by binding and activating NSF [282]. RAB11A is located in recycling endosomes, the Golgi complex and on the cytoplasmic side of cytoplasmic vesicles, and regulates transport from the Golgi to the endosome [283] and from the Golgi to the plasma membrane [283]. RAB11A binds to the neuronal cell adhesion protein L1CAM [284] and therefore is probably involved in its trafficking. The RAB protein RAB14 regulates vesicular transport between the Golgi and early endosomes, and is involved in CDH2 shedding (not shown) [285] and as such affects cell-cell adhesion (not shown) [285]. Lastly, also the ER-shaping protein RTN2 [286] is involved in vesicular ER to Golgi transport [287]. Dysregulated (vesicle) trafficking affects exocytosis, receptor trafficking, (membrane) recycling and ultimately decreases the viability of the neuron.

Mitochondrial function and apoptosis
Mitochondrial dysfunction is associated with both familial and sporadic PD [309]. BCL2, located in the nuclear membrane and in the mitochondrial outer membrane, is an important anti-apoptotic factor that binds to, inhibits and decreases the expression of the proapoptotic protein BAX [310][311][312][313]. BAX inhibition is mediated via the inhibition of MAPK8 that inhibits the binding of YWHAZ and BAX, and in this way increases the translocation of BAX to the mitochondrial membrane [314]. BCL2 is bound and regulated by multiple proteins in the landscape, i.e., SATB1 decreases and NFKBIA increases BCL2 expression [315,316]. MAPK8 also increases BCL2 expression [317], but inhibits BCL2 function [318,319]. BCL2 in turn inhibits MAPK8 [319], decreases expression of NFKBIA[320], NDRG1 [321] and PTEN [322], increases expression of SNAP25 [321], and decreases cleavage of SRPK2 [323]. BCL2 binds MAPK8 [324], CASP3 [325]and PARK2 [326], and inhibits apoptotic pathways in that it, in addition to inhibiting BAX, also inhibits CASP3 [327] and HTRA2 [328] and HTRA2 translocation out of mitochondria [329]. In the cytoplasm, HTRA2 binds EIF4G1 [330], PARK2 (not shown) [331], PINK1 (not shown) [332] and CDK5 [332]. CDK5 in turn inhibits PARK2 [176] and increases TH expression [7]. SNCA binds to PARK2 [333] and, in contrast to CDK5, decreases TH expression [334,166]. Other proteins in the landscape that affect mitochondrial function are MRPL15, ATP5C1 and RET. The 39S ribosomal protein MRPL15 is located in mitochondria and involved in mitochondrial-specific protein expression. Moreover, MRPL15 binds to the transcription factor SOX2 (not shown) [238] and as such may affect DA-neuron-specific expression (see paragraph 'DA neuron signature' in the section 'Transcriptional and translational regulation'). The ATPase ATP5C1 is part of complex V of the respiratory chain that uses the proton gradient across the mitochondrial membrane to produce ATP from ADP [11]. SNCA may also affect the respiratory chain directly by binding to ATP5C1 [159]. Lastly, the tyrosine kinase RET increases the expression of TH and SLC6A3 (Figure 1a), and ameliorates complex I dysfunction in a PD model [335]. Figure 2. Molecular landscape of interacting proteins, encoded by the mRNAs that are differentially expressed in the striatum of both human PD patients and MPTP-treated mice located in the post-synapse of a striatal neuron.

Detailed description of the biological processes depicted in
As a result of the dysregulation of the biological processes constituting the molecular landscape of the processes shared in the SN (summarized in figure 1), the release of DA to the striatum is decreased. Due to the lower DA release into the synaptic cleft, affecting protein expression in the striatal post-synapse, the activation of the DA receptors DRD2 and DRD3 is diminished; these receptors are associated with PD [336,337]. When activated, DRD2 (long variant) and DRD3 increase intracellular calcium [338], but they also inhibit the function of the NMDA receptor(NMDAR) [339] and the VDCC [340,341]. The VDCC binds to ITSN1[39], a protein involved in actin reorganization and assembly [342,343]. DCLK1 and ENC1 are also involved in actin regulation, i.e. DCLK1 regulates the distribution of actin [344] and ENC1 is an actin-binding protein [345] that also binds to SNCA [161]. DRD2 also binds to calmodulin (CaM) [346,347] and thereby exerts influence on calcium signaling in the striatal neuron. Namely, CaM binds to the VDCC [142], the NMDAR (not shown) [348], SNCA [349], LRRK2 [173], TGM2 [350], KCNQ5 [351], DIRAS2 [352] and DCLK1 [352], and can thereby affect multiple proteins in the landscape. Furthermore, CaM regulates KCNQ5 [353] and inhibits calcium flux through the NMDAR into the cell [354,355]. In addition, calcium-bound CaM activates CREB1 [356,357] and CAMK1G [358], and regulates TGM2 function (not shown) [350]. CAMK1G also activates CREB1 [358], and TGM2 activates ERK1/2 [359] and CREB1 [360], but also binds to CASP3 [361], decreases the expression of KCNQ5 [362] and increases the expression of LRRK2 [362]. TGM2 is also activated by calcium [363], increases the efflux of calcium out of the cell [364], binds to SNCA [365] and increases its aggregation (not shown) [365,366]. Calcium and CaM therefore affect the activity of ERK1/2 and CREB1 either directly or via the activation of TGM2 or CAMK1G. Activation of DRD2 by DA also results in the activation of ERK1/2 [367] and CREB1 [368]. ERK1/2 binds to CHGB [369] and the familial proteins SNCA [370] and PARK7 [371]. Furthermore, in addition to DRD2 and TGM2 (see above), S100A10 [372] and ITSN1 [373] activate ERK1/2, whereas the nuclear membrane protein TMEM176B inhibits ERK1/2 activation [374]. Of note, all these processes converge on CREB1. ERK1/2 activates CREB1 [375,376], and CREB1 is activated by CaM, CAMK1G, TGM2 and DRD2 (see above), but also by the NMDAR [356,377] and the (L-type) VDCC (not shown) [356] due to their ability to increase calcium influx, which is necessary for CREB1 activation [378,379]. Thus, CREB1 is regulated by the majority of the proteins in the striatal landscape, either directly or via ERK1/2 activation. Moreover, DA activates both ERK1/2 [367] and CREB1 (via the DA receptors) [380,368], suggesting that ERK1/2 and CREB1 activation (via phosphorylation) is reduced in PD or after MPTP treatment due to the absence of DA.
These pathways also play a role in the effect of L-DOPA, the mainstay of treatment in PD. L-DOPA administration activates ERK1/2 in the striatum [381]. DA-induced, CREB1-dependent transcription in the intact striatum in a PD model [382] is further potentiated by NMDAR activation [377]. The secretory granule protein CHGB is one of the proteins of which the expression is regulated by CREB1, i.e. CREB1 binds to the CRE element of the CHGB gene promoter [383]. Furthermore, calcium decreases the expression of CHGB [384] and CHGB binds to PARK2 [263]. In addition to ERK1/2 and CREB1, L-DOPA also activates DRD2 [ 386], and increases the expression of DRD3 [386], CASP3 [387] and S100A10 [388]. In a PD rat model, S100A10 is involved in L-DOPA-induced abnormal involuntary movements [389]. The activation of striatal ERK1/2 by L-DOPA also appears involved in L-DOPA-induced dyskinesias [389], but not the L-DOPA induced CREB1 activation [390,391,381]. These processes could therefore not only give insights into the PD-related disease mechanisms in the striatum, but also in the beneficial, and adverse, effects of pharmacological treatment. CREB1 and ERK1/2 are also known for their role in epilepsy. Brain areas prone to epileptic seizures show an increased activation of CREB1 and ERK1/2 [382], and an up regulation of CHGB [392], CREB1 [392], ENC1 [356] and NPTX2 [392]. NPTX2 is thought to play a role in long-term plasticity [392] and increases apoptosis [11]. Further, KCNQ5 [393], the NMDAR [394] and the VDCC [395] are associated with epileptic seizures. Therefore, the landscape cannot only give insight in treatment outcome, but can also explain the associations seen in functional studies with PD, in this respect with epilepsy [396].