Effects of dopaminergic cell degeneration on electrophysiological characteristics and GAD65/GAD67 expression in the substantia nigra: different action on GABA cell subpopulations.

The motor disturbances occurring in Parkinson's disease have been partially attributed to a hyperactivity of gamma-aminobutyric acid (GABA)-ergic nigral cells largely in the substantia nigra pars reticulata (SNr) secondary to the degeneration of dopaminergic nigrostriatal neurons. However, some aspects of this response remain unclear. In this work, different electrophysiological and neurochemical parameters were studied in GABAergic cells of the SN after unilateral nigrostriatal dopaminergic lesion using 6-hydroxydopamine injection in rats. Our data showed that 1) the SN under normal conditions contains different subsets of GABAergic cells according to their firing pattern and glutamic acid decarboxylase mRNA levels, and 2) the response of these GABAergic cell subgroups was different after the ipsi- and contralateral dopaminergic cell degeneration. These findings indicate a complex regulation of nigral GABAergic activity after nigrostriatal dopaminergic degeneration that probably involves local mechanisms, the nigro-striato-nigral loop, as well as interhemispheric mechanisms whose anatomical basis remains unstudied.     

Nigral Pathology Contributes to Microstructural Integrity of Striatal and Frontal Tracts in Parkinson's Disease

Background

Motor and cognitive impairment in Parkinson's disease (PD) is associated with dopaminergic dysfunction that stems from substantia nigra (SN) degeneration and concomitant α-synuclein accumulation. Diffusion magnetic resonance imaging (MRI) can detect microstructural alterations of the SN and its tracts to (sub)cortical regions, but their pathological sensitivity is still poorly understood.

Objective

To unravel the pathological substrate(s) underlying microstructural alterations of SN, and its tracts to the dorsal striatum and dorsolateral prefrontal cortex (DLPFC) in PD.

Methods

Combining post-mortem in situ MRI and histopathology, T1-weighted and diffusion MRI, and neuropathological samples of nine PD, six PD with dementia (PDD), five dementia with Lewy bodies (DLB), and 10 control donors were collected. From diffusion MRI, mean diffusivity (MD) and fractional anisotropy (FA) were derived from the SN, and tracts between the SN and caudate nucleus, putamen, and DLPFC. Phosphorylated-Ser129-α-synuclein and tyrosine hydroxylase immunohistochemistry was included to quantify nigral Lewy pathology and dopaminergic degeneration, respectively.

Results

Compared to controls, PD and PDD/DLB showed increased MD of the SN and SN-DLPFC tract, as well as increased FA of the SN-caudate nucleus tract. Both PD and PDD/DLB showed nigral Lewy pathology and dopaminergic loss compared to controls. Increased MD of the SN and FA of SN-caudate nucleus tract were associated with SN dopaminergic loss. Whereas increased MD of the SN-DLPFC tract was associated with increased SN Lewy neurite load.

Conclusions

In PD and PDD/DLB, diffusion MRI captures microstructural alterations of the SN and tracts to the dorsal striatum and DLPFC, which differentially associates with SN dopaminergic degeneration and Lewy neurite pathology. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.     

Central dopamine deficiency in pure autonomic failure

Objective Pure autonomic failure (PAF) and Parkinson’s disease (PD) share several clinical laboratory abnormalities; however, PAF is not associated with parkinsonism. In this study, we tested the hypothesis that preservation of nigrostriatal dopaminergic innervation explains the absence of motor dysfunction in PAF. Methods Patients with PAF (N = 5) or PD (N = 21) and control subjects (N = 14) had brain 6-[¹⁸F]fluorodopa positron emission tomographic scanning and cerebrospinal fluid catechol measurements. A patient with PAF and another with PD had rapid postmortem striatal, nigral, and sympathetic ganglion sampling, with assays of catechols and tyrosine hydroxylase activity. Results The PAF and PD groups had similarly low mean substantia nigra (SN):occipital (OCC) ratios of 6-[¹⁸F]fluorodopa-derived radioactivity and similarly low cerebrospinal fluid dihydroxyphenylacetic acid and DOPA levels. Only the PD group, however, had low PUT:OCC, caudate:OCC, or PUT:SN ratios. The PAF and PD cases had similarly low SN tissue concentrations of dopamine and tyrosine hydroxylase activity, but the PD patient had tenfold lower PUT dopamine and the PAF patient 15-fold lower myocardial norepinephrine concentrations. Conclusions Surprisingly, PAF and PD entail similarly severe nigral and overall central dopaminergic denervation. There is more severe loss of striatal dopaminergic terminals in PD than in PAF and more severe loss of sympathetic noradrenergic terminals in PAF than in PD. These differences explain the distinctive clinical manifestations of the two Lewy body diseases. Parkinsonism appears to reflect striatal dopamine deficiency rather than loss of nigral dopaminergic neurons per se.     

Striatal and nigral COX-2 expression after chronic typical and atypical neuroleptic administration in rats

Haloperidol, but not clozapine, induces dopaminergic nigrostriatal degeneration. However, the mechanisms by which haloperidol causes neurotoxicity are not fully understood. An increase in cyclooxygenase-2 (COX-2) expression has been observed correlated with nigrostriatal degeneration. We investigated the modifications of striatal and nigral COX-2 expression induced by chronic haloperidol and clozapine administration. Rats were treated for 21 days with: haloperidol (1 mg/kg), clozapine (1 mg/kg) or saline. No significant differences were observed in striatal and nigral COX-2 expression between haloperidol and clozapine-treated animals. This observation might suggest that nigral COX-2 expression is not the underlying mechanisms involved in haloperidol-induced dopaminergic neurodegeneration.     

Transcranial sonography in the early and differential diagnosis of Parkinson's disease

In recent years transcranial sonography (TCS) has become a widely used method for the visualization of the brain parenchyma through the intact scull. Using TCS, our group discovered changes of the echotexture--namely increased echogenicity--at the substantia nigra (SN) in about 90% of patients with Parkinson's disease (PD). These results assessed with an interrater reproducibility of r = 0.8 in several studies have been confirmed by several other groups. In contrast increased SN echogenicity is rarely found in patients with atypical or symptomatic Parkinsonian syndromes, providing a valuable tool for differential diagnosis. Interestingly, increased SN echogenicity can also be found in about 8 to 10% of healthy subjects. In PET analyses more than 60% of these clinically healthy individuals show a subclinical reduction of the striatal 18F-Dopa uptake indicating an alteration of the dopaminergic nigrostriatal system and nigral cell loss. Furthermore, it was possible to demonstrate that this ultrasound finding has a functional impact as subjects with an increased echogenicity of the SN (i) showed more frequently clinical symptoms of asymmetric hypokinesia with increasing age and (ii) developed more often and more severe Parkinsonian side effects when treated with neuroleptic therapy for neuropsychiatric disorders. Longitudinal studies indicate that the ultrasound signal does not change in the course of the disease. Moreover, presymptomatic carriers of mutations causative for monogenetic PD display the same echofeature as their relatives already affected by the disease. These findings indicate that increased SN echogenicity constitutes a biomarker for vulnerability of the nigrostriatal system in healthy subjects and eventually PD in a subgroup of persons with additional risk factors.     

Nigral degeneration in Parkinson's disease in relation to clinical features

Neuronal loss in the substantia nigra (SN) in Parkinson's disease (PD) shows a topographical organisation where the lateral part of the SN is more affected. This is - due to projection of the lateral SN mainly to the putamen - reflected in more complete loss of dopamine content in the putamen than in the caudate nucleus. Of the parkinsonian symptoms rigidity and hypokinesia are associated with neuronal loss in the lateral substantia nigra and the resulting dopamine loss in the putamen. Neuronal mechanisms other than degeneration of the nigrostriatal system seem to be involved in the pathophysiology of tremor.
Cognitive impairment and dementia in PD is related to dysfunction of the cortical cholinergic system, especially in the frontal cortex. The brain dopaminergic system, however, contributes as a subcortical component to cognitive impairment in PD.
Clinical studies have shown that selegiline may slow down the progression of PD. We studied postmortem samples of patients treated with selegiline and levodopa and those with levodopa alone. The number of medial nigral neurons was significantly higher in the selegiline group.
Treatment with selegiline might retard the death of nigral neurons, but further studies are needed to confirm the preliminary findings.     

Adaptive changes in the nigrostriatal pathway in response to increased 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurodegeneration in the mouse

Although several adaptive mechanisms have been identified that mask the existence of Parkinson's disease and delay the onset and aggravation of motor symptoms, the timescale and implications of this compensatory process remain an enigma. In order to examine: (i) the nature of the dopaminergic adaptive mechanisms that come into action; (ii) their sequential activation in relation to the severity of degeneration; and (iii) their efficacy with regard to the maintenance of a normal level of basal ganglia activity, we analysed the brains of mice treated daily with 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP, 4 mg/kg, i.p.) and killed at 5-day intervals from day 0 (D0) to D20. Our results demonstrate the sequential activation of two compensatory mechanisms: (i) an increase in striatal tyrosine hydroxylase (TH) protein content attested by the persistence of TH immunolabelling up to D15, contrasting with the decrease observed in both the number of nigral TH-immunoreactive neurons (-70.2%) and striatal dopamine content (-38.4%); (ii) a downregulation of DA uptake in surviving terminals at D20 (73.4% of nigral degeneration). At this point, the failure of adaptive mechanisms to maintain striatal dopaminergic homeostasis is also illustrated by an increase in the cytochrome oxidase activity of substantia nigra pars reticulata, a marker of neuronal function. It has been postulated that an increase in dopamine release per pulse could constitute an adaptive mechanism. The data we present from our MPTP mice model infirm this hypothesis. This study explores the link between the degree of nigral degeneration and the sequential activation of dopaminergic compensatory mechanisms in the nigrostriatal pathway and, in so doing, proposes a rethink of the paradigm applied to these mechanisms.     

Trisialoganglioside GT1b induces in vivo degeneration of nigral dopaminergic neurons: role of microglia

We recently showed that trisialoganglioside (GT1b) induces cell death of dopaminergic neurons in rat mesencephalic cultures (Chung et al., Neuroreport 12:611-614, 2001). The present study examines the in vivo neurotoxic effects of GT1b on dopaminergic neurons in the substantia nigra (SN) of Sprague-Dawley rats. Seven days after GT1b injection into the SN, immunocytochemical staining of SN tissue revealed death of nigral neurons, including dopaminergic neurons. Additional immunostaining using OX-42 and OX-6 antibodies showed that GT1b-activated microglia were present in the SN where degeneration of nigral neurons was found. Western blot analysis and double-labeled immunohistochemistry showed that inducible nitric oxide synthase (iNOS) was expressed in the SN, where its levels were maximal at 8 h post-GT1b injection, and that iNOS was localized exclusively within microglia. GT1b-induced loss of dopaminergic neurons in the SN was partially inhibited by N(G)-nitro-L-arginine methyl ester hydrochloride, an NOS inhibitor. Our results indicate that in vivo neurotoxicity of GT1b against nigral dopaminergic neurons is at least in part mediated by nitric oxide released from activated microglia. Because GT1b exists abundantly in central nervous system neuronal membranes, our data support the hypothesis that immune-mediated events triggered by endogenous compounds such as GT1b could contribute to the initiation and/or the progression of dopaminergic neuronal cell death that occurs in Parkinson's disease.     

L-alpha-aminoadipic acid restricts dopaminergic neurodegeneration and motor deficits in an inflammatory model of Parkinson’s disease in male rats

Neuroinflammation is a contributory factor underlying the progressive nature of dopaminergic neuronal loss within the substantia nigra (SN) of Parkinson's disease (PD) patients, albeit the role of astrocytes in this process has been relatively unexplored to date. Here, we aimed to investigate the impact of midbrain astrocytic dysfunction in the pathophysiology of intra-nigral lipopolysaccharide (LPS)-induced experimental Parkinsonism in male Wistar rats via simultaneous co-injection of the astrocytic toxin L-alpha-aminoadipic acid (L-AAA). Simultaneous intra-nigral injection of L-AAA attenuated the LPS-induced loss of tyrosine hydroxylase-positive (TH⁺) dopamine neurons in the SNpc and suppressed the affiliated degeneration of TH⁺ dopaminergic nerve terminals in the striatum. L-AAA also repressed LPS-induced nigrostriatal dopamine depletion and provided partial protection against ensuing motor dysfunction. L-AAA abrogated intra-nigral LPS-induced glial fibrillary acidic protein-positive (GFAP⁺) reactive astrogliosis and attenuated the LPS-mediated increases in nigral S100β expression levels in a time-dependent manner, findings which were associated with reduced ionized calcium binding adaptor molecule 1-positive (Iba1⁺) microgliosis, thus indicating a role for reactive astrocytes in sustaining microglial activation at the interface of dopaminergic neuronal loss in response to an immune stimulus. These results indicate that midbrain astrocytic dysfunction restricts the development of dopaminergic neuropathology and motor impairments in rats, highlighting reactive astrocytes as key contributors in inflammatory associated degeneration of the nigrostriatal tract.     

The acute and the long-term effects of nigral lipopolysaccharide administration on dopaminergic dysfunction and glial cell activation

Sustained reactive microgliosis may contribute to the progressive degeneration of nigral dopaminergic neurons in Parkinson's disease (PD), in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposed human and in non-human primates. However, the temporal relationship between glial cell activation and nigral cell death is relatively unexplored. Consequently, the effects of acute (24 h) and chronic (30 days) glial cell activation induced by unilateral supranigral lipopolysaccharide (LPS) administration were studied in rats. At 24 h, LPS administration caused a marked reduction in the number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra (SN) but striatal TH-ir was unaffected. By 30 days, the loss of TH-positive neurons in the LPS-treated nigra was no greater than at 24 h although a heterogeneous loss of striatal TH-ir was present. The loss of nigrostriatal neurons was of functional significance, as at 30 days, LPS-treated rats exhibited ipsiversive circling in response to (+)-amphetamine administration. At 24 h, there was a moderate increase in glial fibrillary acidic protein (GFAP)-ir astrocytes in the SN but a marked elevation of p47phox positive OX-42-ir microglia, and intense inducible nitric oxide synthase (iNOS)-ir and 3-nitrotyrosine (3-NT)-ir was present. However, by 30 days the morphology of OX-42-ir microglia returned to a resting state, the numbers were greatly reduced and no 3-NT-ir was present. At 30 days, GFAP-ir astrocytes were markedly increased in number and iNOS-ir was present in fibrillar astrocyte-like cells. This study shows that acute glial activation leading to dopaminergic neuron degeneration is an acute short-lasting response that does not itself perpetuate cell death or lead to prolonged microglial activation.