A holistic rat model to investigate therapeutic interventions in Parkinson's disease: viral induction of a slow-progressing motor phenotype, dopaminergic degeneration and early microglia neuroinflammation.

Delayed Dominant-Negative TNF Gene Therapy Halts Progressive Loss of Nigral Dopaminergic Neurons in a Rat Model of Parkinson's Disease

Ventral midbrain dopaminergic neurons: From neurogenesis to neurodegeneration

Pesticide Exposure Exacerbates α-Synucleinopathy in an A53T Transgenic Mouse Model

2021 Workshop: Neurodegenerative Diseases in the Gut-Brain Axis—Parkinson's Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease among the elderly. While sporadic PD constitutes 99% of the cases, the remaining 1% is of genetic origin. The neuropathological hallmarks of PD are progressive degeneration of dopaminergic (DA) neurons and presence of Lewy neurites and Lewy bodies (LBs) intracytoplasmic proteinaceous inclusions that contain -synuclein (SYN), synphilin-1, components of the ubiquitin proteasomal pathway and parkin (Dawson, 2006). The loss of DA neurons in substantia nigra pars compacta (SNpc) results in decreased signalling in the striatum thereby giving rise to motor defects like resting tremor, bradykinesia, rigidity and posture instability. Besides DA neuronal loss, microglial activation and increased astroglial and lymphocyte infiltration also occur in PD. A role for inflammation in PD has been inferred from the identification of human leukocyte antigen (HLA)-DR positive reactive microglia in the brains of PD patients (McGeer et al., 1988). Additionally, levels of proinflammatory cytokines like IL-6, IL-1, TNF have been found to be elevated in the blood and cerebrospinal fluid (CSF) of PD patients (Nagatsu & Sawada, 2005; Dawson, 2006) Although these inflammatory components might serve as useful biomarkers, the aetiology of striatal DA degeneration still remains enigmatic. In the last decade, identification of mutations in several distinct genes (LRRK2, parkin, PINK1, DJ-1, -synuclein, MAPT, UCHL1 etc) linked to different forms of familial Parkinsonism has imparted a new direction to understanding PD pathogenesis (Tong & Shen, 2009). The question as to how seemingly divergent genes cause PD still remains unanswered, as there is no common molecular pathway involving these gene products. While parkin, -synuclein (SYN) and ubiquitin C-terminal hydrolase L1 (UCHL1) are functionally associated with the cellular ubiquitin proteasomal system (UPS), DJ-1 and PINK1 protect against oxidative stress and mitochondrial dysfunction. More recently, microarray analysis of SNpc from parkinsonian brain (Mandel et al., 2005) has shown that 68 genes related to protein degradation, signal transduction, dopaminergic transmission, iron transport and glycolysis are downregulated. Prominent among these are the protein chaperone HSC-70, subunits of the UPS and SKP1A, a member of the E3 ubiquitin ligase complex. Therefore, it is most likely that impairment in energy metabolism and/or alterations in UPS are the underlying mechanisms for PD pathogenesis (Eriksen et al., 2005; Mandel et al., 2005). Current PD treatment regimes can be divided into three categories: symptomatic, protective and restorative. Only symptomatic treatment via the administration of L-dopa and other

Dopamine quinone toxicity has been implicated in the degeneration of nigral dopaminergic (DA) neurons in Parkinson’s disease (PD). NAD(P)H:quinone oxidoreductase (NQO1) may protect against this quinone toxicity. In Parkinsonian brains, levels of NQO1 are increased in reactive glia cells that are located around the remaining DA neurons in the substantia nigra pars compacta (SNc), suggesting a neuroprotective role of NQO1.It is not known at which stage of the disease process the upregulation of glial NQO1 starts. Furthermore, it is at present not clear whether NQO1 indeed plays a neuroprotective role in the disease process. As a first step to experimentally study a potential neuroprotective role of NQO1, it was examined whether activation of glia cells and changes in the distribution of NQO1-positive glia cells and the expression levels of glial NQO1, as seen in PD brains, also occurred in a 6-hydroxydopamine (6-OHDA) rat model of PD.Our results show that astroglia cells and microglia cells were activated. Furthermore, NQO1 was upregulated in astroglia cells in the SNc in those areas in which DA neurons degenerated. The time course and pattern of upregulation of NQO1 paralleled those of the degeneration of DA neurons. Activated microglia were seen at a later stage during the course of degeneration of DA neurons.In conclusion, in the present model, astroglia cells and microglia cells are activated in response to 6-OHDA-induced oxidative stress. Furthermore, levels of NQO1 are increased in astroglia cells. The findings in the present model are in line with the findings as seen in parkinsonian brains. The 6-OHDA rat model of PD is, therefore, suitable for further research to examine a potential neuroprotective role of NQO1.KeywordsTyrosine HydroxylaseAstroglia CellGlia CellGFAP ImmunoreactivityParkinsonian BrainThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

We have recently identified a neuroprotective role for omega-3 polyunsaturated fatty acids (n-3 PUFAs) in a toxin-induced mouse model of Parkinson's disease (PD). Combined with epidemiological data, these observations suggest that low n-3 PUFA intake is a modifiable environmental risk factor for PD. In order to strengthen these preclinical findings as prerequisite to clinical trials, we further investigated the neuroprotective role of n-3 PUFAs in Fat-1 mice, a transgenic model expressing an n-3 fatty acid desaturase converting n-6 PUFAs into n-3 PUFAs. Here, we report that the expression of the fat-1 transgene increased cortical n-3:n-6 PUFA ratio (+28%), but to a lesser extent than dietary supplementation (92%). Such a limited endogenous production of n-3 PUFAs in the Fat-1 mouse was insufficient to confer neuroprotection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity as assessed by dopamine levels, tyrosine hydroxylase (TH)-positive neurons and fibers, as well as nigral Nurr1 and dopamine transporter (DAT) mRNA expression. Nevertheless, higher cortical docosahexaenoic acid (DHA) concentrations were positively correlated with markers of nigral dopaminergic neurons such as the number of TH-positive cells, in addition to Nurr1 and DAT mRNA levels. These associations are consistent with the protective role of DHA in a mouse model of PD. Taken together, these data suggest that dietary intake of a preformed DHA supplement is more effective in reaching the brain and achieving neuroprotection in an animal model of PD.

HSP70 and Constitutively Active HSF1 Mediate Protection Against CDCrel-1-mediated Toxicity

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Accepted September 14, 1998. From the Neuropharmacology Unit, Defense and Veterans Head Injury Program, Henry M. Jackson Foundation, and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland; Veterans Administration Medical Center GRECC, Bedford, Massachusetts; and Departments of Neurology and Pathology, Boston University Medical School, Boston, Massachusetts. Address correspondence to Dr. Litvan, Neuropharmacology Unit, Defense and Veterans Head Injury Program, Henry M. Jackson Foundation, NINDS, NIH, Federal Building, Room 714, 7550 Wisconsin Avenue, Bethesda, MD 20892-9130; e-mail: litvan1@helix.nih.gov Copyright q 1999 American Psychiatric Press, Inc. Clinicopathologic Case Report

Animal models of human pathologies remain invaluable tools for unraveling disease mechanisms and evaluating potential therapeutic strategies. For a number of diseases, the lack of a reliable animal model represents an important limiting step towards the development of efficient treatments. This holds particularly true for Parkinson's disease (PD), a major neurodegenerative disorder for which only symptomatic treatments currently exist. The difficulties encountered by researchers to reproduce PD pathology in animals stem primarily from an incomplete understanding of the disease. Indeed, the cause of the disease remains unknown in 90% of cases, referred to as sporadic or idiopathic. The discovery of familial forms of the disease, however, has led to the development of a large number of transgenic mice models based on genetic modifications that play a direct causative role in a significant proportion of human PD cases. Unfortunately, these transgenic mice fail to recapitulate the robust neurodegeneration of dopaminergic (DAergic) neurons of the substantia nigra pars compacta (SNpc) and concomitant loss of DAergic projections to the striatum, the neuropathological hallmark of the human condition. The lack of nigral pathology severely limits the usefulness of such models for pre-clinical evaluation of potential therapeutics. Viral vector gene delivery tools represent an interesting alternative to classical transgenesis as they allow for targeted and high-level transgene expression in the nigrostriatal system of adult animals. During the course of this thesis we have developed two new viral vector-based rodent models of PD. In our first model, we have used a recombinant adeno-associated virus (rAAV) vector, with a high tropism towards nigral DAergic neurons, to drive overexpression of the parkin-associated endothelin receptor-like receptor (Pael-R) in the SNpc of adult rats. Indeed, accumulation of Pael-R is implicated in the pathogenesis of autosomal-recessive juvenile parkinsonism (AR-JP), a young-onset familial form of PD. We show that insoluble accumulation of Pael-R in rats induces a rapidly progressing degeneration of nigral DAergic neurons and a loss of DAergic fibers and terminals in the striatum. Lesioned animals also displayed spontaneous behavioral abnormalities linked to depletion of striatal dopamine (DA) and persisting up to 6 months post-injection. Chronic accumulation of Pael-R in the nigrostriatal system of adult rats therefore represents a robust and highly reproducible model of PD, recapitulating key pathological and phenotypical features of the human condition. The second model developed was based on nigral delivery of the PD-associated mutant G2019S leucine-rich repeat kinase 2 (LRRK2) protein. Indeed, the G2019S mutation in the LRRK2 gene is the most important genetic determinant of PD, accounting for a significant proportion of both familial and sporadic PD cases. Due to the large size of the LRRK2 coding sequence, an adenoviral system with a high packaging capacity was used to drive expression of the protein. Recombinant adenoviral (rAd) vectors are potentially pro-inflammatory and less efficient tools as rAAV vectors for long-term gene delivery to the SNpc. Nevertheless, through retrograde axonal delivery of rAd-LRRK2 particles, we achieved robust and neuron-specific expression of full-length wild-type or mutant G2019S human LRRK2 in nigral DAergic neurons of adult rats. Expression persisted up to 6 weeks post-injection, with no visible signs of inflammation in the SNpc. We demonstrate that the wild-type form of LRRK2 does not induce neuronal loss when expressed in the SNpc. In contrast, under the same conditions and levels of expression as the wild-type form, the PD-associated G2019S mutation in LRRK2 is sufficient to cause a progressive loss of nigral dopaminergic neurons. This is the first demonstration of frank dopaminergic neuronal degeneration in rodents induced by the expression of G2019S mutant LRRK2. Our data also provide a new rodent model of LRRK2-linked PD which recapitulates one of the cardinal pathological features of the disease. In the absence of a clear understanding of human PD pathogenesis, the development of multiple transgenic models may help to identify common disease mechanisms and drug targets. A potential treatment identified in one model may be effective only in the corresponding subset of PD patients, carrying this particular genetic modification. On the other hand, the pathogenic pathway targeted may also be activated in sporadic PD. Ultimately, whether or not these models stand the test of time will depend on how effective newly-identified drugs will be, not only on AR-JP or LRRK2-linked PD patients, but above all on sporadic PD, which represents the vast majority of PD patients.

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting approximately 1% of the population older than 60 years. Classically, PD is considered to be a motor system disease and its diagnosis is based on the presence of a set of cardinal motor signs (rigidity, bradykinesia, rest tremor) that are consequence of a pronounced death of dopaminergic neurons in the substantia nigra pars compacta. Nowadays there is considerable evidence showing that non-dopaminergic degeneration also occurs in other brain areas which seems to be responsible for the deficits in olfactory, emotional and memory functions that precede the classical motor symptoms in PD. The present review attempts to examine results reported in epidemiological, clinical and animal studies to provide a comprehensive picture of the antiparkinsonian potential of caffeine. Convergent epidemiological and pre-clinical data suggest that caffeine may confer neuroprotection against the underlying dopaminergic neuron degeneration, and influence the onset and progression of PD. The available data also suggest that caffeine can improve the motor deficits of PD and that adenosine A2A receptor antagonists such as istradefylline reduces OFF time and dyskinesia associated with standard 'dopamine replacement' treatments. Finally, recent experimental findings have indicated the potential of caffeine in the management of non-motor symptoms of PD, which do not improve with the current dopaminergic drugs. Altogether, the studies reviewed provide strong evidence that caffeine may represent a promising therapeutic tool in PD, thus being the first compound to restore both motor and non-motor early symptoms of PD together with its neuroprotective potential.

Parkinson's disease (PD) is a progressive neurodegenerative brain disease due to degeneration of dopaminergic neurons (DNs) presented with motor and non-motor symptoms. PD symptoms are developed in response to the disturbance of diverse neurotransmitters including γ-aminobutyric acid (GABA). GABA has a neuroprotective effect against PD neuropathology by protecting DNs in the substantia nigra pars compacta (SNpc). It has been shown that the degeneration of GABAergic neurons is linked with the degeneration of DNs and the progression of motor and non-motor PD symptoms. GABA neurotransmission is a necessary pathway for normal sleep patterns, thus deregulation of GABAergic neurotransmission in PD could be the potential cause of sleep disorders in PD. Sleep disorders affect GABA neurotransmission leading to memory and cognitive dysfunction in PD. For example, insomnia and short sleep duration are associated with a reduction of brain GABA levels. Moreover, PD-related disorders including rigidity and nocturia influence sleep patterns leading to fragmented sleep which may also affect PD neuropathology. However, the mechanistic role of GABA in PD neuropathology regarding motor and non-motor symptoms is not fully elucidated. Therefore, this narrative review aims to clarify the mechanistic role of GABA in PD neuropathology mainly in sleep disorders, and how good GABA improves PD. In addition, this review of published articles tries to elucidate how sleep disorders such as insomnia and REM sleep behavior disorder (RBD) affect PD neuropathology and severity. The present review has many limitations including the paucity of prospective studies and most findings are taken from observational and preclinical studies. GABA involvement in the pathogenesis of PD has been recently discussed by recent studies. Therefore, future prospective studies regarding the use of GABA agonists in the management of PD are suggested to observe their distinct effects on motor and non-motor symptoms. There is a bidirectional relationship between the pathogenesis of PD and sleep disorders which might be due to GABA deregulation.

The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled vector. SKP1A silencing resulted in increased susceptibility to neuronal damages induced by the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium ion and serum starvation, in parallel with a decline in the expression of the dopaminergic markers, dopamine transporter and vesicular monoamine transporter-2. SKP1A-deficient cells presented a delay in completion of the cell cycle and the inability to arrest at the G(0)/G(1) phase when induced to differentiate. Instead, the cells progressed through S phase, developing rounded aggregates with characteristics of aggresomes including immunoreactivity for gamma-tubulin, alpha-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 (70-kDa heat shock cognate protein), and proteasome subunit, and culminating in a lethal phenotype. Conversely, stably enforced expression of wild type SKP1A duplicated the survival index of naïve SN4741 cells under proteasomal inhibition injury, suggesting a new structural role of SKP1 in dopaminergic neuronal function, besides its E3 ligase activity. These results link, for the first time, SKP1 to dopamine neuronal function and survival, suggesting an essential role in sporadic PD. In summary, this new model has reproduced to a significant extent the molecular alterations described in sporadic PD at the cellular level, implicating Skp1 as a potential modifier in sporadic PD neurodegeneration.