LRRK2 expression in idiopathic and G2019S positive Parkinson's disease subjects: a morphological and quantitative study.

AIMS: Mutations in the gene encoding leucine-rich repeat kinase-2 (LRRK2) have been established as a common genetic cause of Parkinson's disease (PD). The distribution of LRRK2 mRNA and protein in the human brain has previously been described, although it has not been reported in PD cases with the common LRRK2 G2019S mutation. METHODS: To further elucidate the role of LRRK2 in PD, we determined the localization of LRRK2 mRNA and protein in post-mortem brain tissue from control, idiopathic PD (IPD) and G2019S positive PD cases. RESULTS: Widespread neuronal expression of LRRK2 mRNA and protein was recorded and no difference was observed in the morphological localization of LRRK2 mRNA or protein between control, IPD and G2019S positive PD cases. Using quantitative real-time polymerase chain reaction, we demonstrated that there is no regional variation in LRRK2 mRNA in normal human brain, but we have identified differential expression of LRRK2 mRNA with significant reductions recorded in limbic and neocortical regions of IPD cases compared with controls. Semi-quantitative analysis of LRRK2 immunohistochemical staining demonstrated regional variation in staining intensity, with weak LRRK2 immunoreactivity consistently recorded in the striatum and substantia nigra. No clear differences were identified in LRRK2 immunoreactivity between control, IPD and G2019S positive PD cases. LRRK2 protein was identified in a small proportion of Lewy bodies. CONCLUSIONS: Our data suggest that widespread dysregulation of LRRK2 mRNA expression may contribute to the pathogenesis of IPD.

Evidence of a breakdown of corticostriatal connections in Parkinson's disease.

Dendritic spines are important structures which receive synaptic inputs in many regions of the CNS. The goal of this study was to test the hypothesis that numbers of dendritic spines are significantly reduced on spiny neurones in basal ganglia regions in Parkinson's disease as we had shown them to be in a rat model of the disease [Exp Brain Res 93 (1993) 17]. Postmortem tissue from the caudate and putamen of patients suffering from Parkinson's disease was compared with that from people of a similar age who had no neurological damage. The morphology of Golgi-impregnated projection neurones (medium-sized spiny neurones) was examined quantitatively. The numerical density of dendritic spines on dendrites was reduced by about 27% in both nuclei. The size of the dendritic trees of these neurones was also significantly reduced in the caudate nucleus from the brains of PD cases and their complexity was changed in both the caudate nucleus and the putamen. Dendritic spines receive crucial excitatory input from the cerebral cortex. Reduction in both the density of spines and the total length of the remaining dendrites is likely to have a grave impact on the ability of these neurones to function normally and may partly explain the symptoms of the disorder.

Deficits in a tricarboxylic acid cycle enzyme in brains from patients with Parkinson's disease.

Parkinson's disease (PD) is associated with mitochondrial dysfunction, specifically a deficiency of complex I of the electron transport chain. Most, although not all, studies indicate that this deficiency is limited to brain regions with neurodegeneration. The current studies tested for deficiencies in other mitochondrial components in PD brain in a neuropathologically unaffected region where the abnormality cannot be attributed to secondary effects of neurodegeneration. The activity of a key (and arguably rate-limiting) tricarboxylic acid cycle enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), was measured in the cerebellum of patients with PD. Activity in 19 PD brains was 50.5% of that in 18 controls matched for age, sex, post-mortem interval, and method of preservation (P<0.0019). The protein subunits of KGDHC were present in normal amounts in PD brains, indicating a relatively discrete abnormality in the enzyme. The activities of another mitochondrial enzyme, glutamate dehydrogenase (GDH), were normal in PD brains. These results demonstrate that specific reductions in KGDHC occur even in pathologically unaffected areas in PD, where the decline is unlikely to be a non-specific result of neurodegeneration. Reductions in the activity of this enzyme, if widespread in the brain, may predispose vulnerable regions to further damage.

Metabolic enzyme expression in dopaminergic neurons in Parkinson's disease: an in situ hybridization study.

To clarify the role of neuronal complex 1 activity in idiopathic Parkinson's disease (IPD), expression of mitochondrial mRNA encoding the ND1 subunit of mitochondrial complex I was examined by semiquantitative in situ hybridization histochemistry in melanized neurons of human substantia nigra in IPD cases and control subjects. Expression of mRNA encoding the glycolytic enzyme, aldolase C, was also examined in substantia nigra and other neurons of the midbrain and brain stem. ND1 mRNA expression was strong in melanized substantia nigra neurons but undetectable in nigral glia. Levels of expression in nigral neurons were higher than in neurons of the red nucleus or cranial nerve nuclei, but similar values were obtained in pontine neurons. ND1 mRNA expression was reduced by about 25% in melanized neurons in IPD. There was no relationship between ND1 expression per cell and disease duration or L-DOPA dosage in the IPD group. No change in ND1 expression was observed in pontine neurons in IPD, and ND1 expression in the locus ceruleus was also unchanged. Melanized nigral neurons expressed lower levels of aldolase C mRNA than other midbrain or brain stem populations in both control and IPD material. These findings suggest that dopamine neurons are more strongly dependent on mitochondrial energy metabolism and oxidative phosphorylation than other brain stem populations. Because mitochondrial complex I activity is significantly reduced in IPD, intrinsically low expression of glycolytic enzymes, together with disease-related reduction in complex I activity, may be a contributory factor predisposing nigral neurons

Up-regulation of tyrosine hydroxylase mRNA in a sub-population of A10 dopamine neurons in Parkinson's disease.

Neuronal injury has been consistently found in A10 midbrain dopamine neurons in Parkinson's disease (PD). To assess changes in neurotransmitter-related gene transcription, in these neurons in PD, tyrosine hydroxylase (TH) mRNA expression was examined in the ventral tegmental area (VTA) of seven PD cases and seven control subjects, using in situ hybridization histochemistry (ISHH). In controls, TH mRNA expression was found in both melanised and non-melanised neurons in the VTA. Neither population expressed dopamine beta-hydroxylase (DBH). Of the melanised neurons, 99% were TH mRNA positive. The level of the TH mRNA signal (expressed as grain density per cell) was similar in the two populations (melanised: 0.129+/-0.004 (mean+/-S.E.M.), n=142 vs. non-melanised: 0.138+/-0.006, n=89, P>0.05, Student's t-Test). In PD cases there was no significant change in TH mRNA expression in melanised neurons (0.138+/-0.003, n=196), and the proportion of positively labeled melanised neurons was 98%. However, non-melanised neurons showed significantly higher TH mRNA (0.163+/-0.006, n=87) than non-melanised neurons in control subjects (P<0.005) and melanised neurons in the PD cases (P<0.0005). This up-regulation of TH mRNA expression in non-melanised neurons may suggest the existence of a compensatory mechanism at presynaptic level.

NMDA NR1 subunit mRNA and glutamate NMDA-sensitive binding are differentially affected in the striatum and pre-frontal cortex of Parkinson's disease patients.

Changes in the levels of mRNA for the NR1 subunit of the glutamate NMDA receptor and in NMDA-sensitive glutamate binding were investigated in consecutive sections of the prefrontal cortex and striatum of control and Parkinson's disease (PD) post-mortem brain using in-situ hybridisation and receptor autoradiography. Both markers of NMDA receptors were found to be relatively unaffected when measured by microdensitometry in the prefrontal cortex of control and PD brains. At a cellular level, a subpopulation of small and medium neurons in the superficial layers of the prefrontal cortex of the PD group showed a decreased expression of NMDA NR1 mRNA, with the maximal decrease in cortical layer IV. In the striatum, levels of glutamate binding to the NMDA receptor detected by receptor autoradiodgraphy were significantly reduced in the PD group, while no change could be detected at a macroscopical level in NMDA NR1 mRNA expression. Consequently, we suggest that the important decrease in agonist binding to the NMDA receptor observed in this study in the caudate and putamen of PD brains, in the absence of any major change in NMDA NR1 mRNA levels might reflect the degeneration of pre-synaptic NMDA receptors located on nigro-striatal projections particularly affected by the disease. Small changes observed at a cellular level in subsets of neurons of both prefrontal cortex and striatum will be discussed at the light of neurochemical changes characteristics of PD.

The vulnerability of nigral neurons to Parkinson's disease is unrelated to their intrinsic capacity for dopamine synthesis: an in situ hybridization study.

The contribution of the dopamine-synthetic capacity of nigral neuronal subregions to their vulnerability to degeneration in idiopathic Parkinson's disease (IPD) was explored using semiquantitative in situ hybridization to study expression of mRNA encoding the rate-limiting dopamine synthetic enzyme, tyrosine hydroxylase (TH). Expression of mRNA, the structural protein, beta-tubulin, and the glycolytic enzyme, fructose-1,6, biphosphate aldolase (aldolase C) was studied in parallel in individual neurons of the substantia nigra pars compacta (SNc) in matched groups of IPD and control subjects. TH mRNA expression was found to be heterogeneously expressed in nigral neurons in control and IPD subjects. There was no significant difference in mean values for TH mRNA expression between control and IPD cases and none between nigral subregions, either in control subjects or in established IPD subjects in this study, but there was evidence for a selective upregulation of TH mRNA expression in non-melanized neurons in IPD. There was no relationship between TH mRNA expression disease duration or L-dopa dosage in the IPD group. Mean TH mRNA values for two additional 40-year-old control subjects fell within the range of values of the aged-control group. Aldolase C and beta-tubulin expression did not differ between control and IPD groups or between nigral subregions. These findings suggest that regulation of dopamine synthesis at the level of the cell body does not play a part in determining the pattern of nigral cell vulnerability in IPD. The heterogeneous pattern of TH synthesis was not age-dependent and may be of physiological significance in nigral function. There was no evidence for compensatory upregulation of TH synthesis in surviving melanized neurons in IPD but non-melanized neurons may be involved in this process. Surviving nigral neurons in IPD appear to retain the capacity for normal aldolase C and beta-tubulin peptide synthesis. Long-term L-dopa treatment does not appear to compromise normal function of nigral dopaminergic neurons.

Basal ganglia neuronal nitric oxide synthase mRNA expression in Parkinson's disease.

Expression of nitric oxide synthase (NOS) mRNA in post mortem brain was studied in putamen, globus pallidus and subthalamic nucleus (STN) of neurologically normal control subjects and patients with Parkinson's disease (PD) using in situ hybridization histochemistry. In PD, a significant increase in NOS mRNA expression was observed in the dorsal two-thirds of the STN with respect to the ventral one-third of the STN. A significant increase in NOS mRNA expression per cell in the medial medullary lamina of the globus pallidus was also observed in PD. NOS mRNA expression was significantly reduced in PD putamen. These findings provide evidence of increased activity of STN neurotransmitter systems in PD and demonstrate for the first time in any species that basal ganglia nitric oxide systems can be selectively regulated in response to changes in dopaminergic input.

DNA fragmentation in human substantia nigra: apoptosis or perimortem effect?

DNA fragmentation was examined in situ in flash-frozen human postmortem midbrain as a marker for programmed cell death. A large series of cases comprising 16 pathologically confirmed idiopathic Parkinson's disease (IPD) cases, 14 control cases without brain pathology, and a group of 6 patients with other parkinsonian movement disorders were examined using TdT-mediated dUTP-biotin 3' end-labeling histology. Labeling of neurons and glia was seen in the substantia nigra of control and IPD cases and in other movement disorder cases. Labeled nuclei were seen in melanized nigral neurons; apoptotic bodies were also found but were more commonly associated with nigral glia. In the control group, labeling of neurons and glia was strongly associated with poor agonal status, assessed by tissue pH, a marker for antemortem hypoxia. The mean tissue pH of the control group with neuronal labeling was 6.28 (SEM .057), which was significantly different from that of the unlabeled group 6.55 (SEM .055). Mean tissue pH for all cases was 6.38. There was no association of nigral neuronal labeling with poor agonal status in the IPD cases, which showed labeling throughout the range of pH values. However, extranigral labeling, seen in the mesencephalon, red nucleus, superior colliculus, rostral pons, and periaqueductal gray matter, in all three subject groups was associated with tissue pH values of less than 6.3. These findings suggest that DNA fragmentation is influenced by antemortem hypoxia and that apoptosis-like changes seen in the postmortem nigra may parallel those seen in experimental ischemia in the animal brain. The likely influence of perimortem factors on these changes indicates that results from postmortem studies of apoptotic cell death in neurodegenerative disease should be treated with caution and underlines the importance of determining postmortem markers for agonal status in human brain.

Selective increase in somatostatin mRNA expression in human basal ganglia in Parkinson's disease.

Levels of the neurotransmitter somatostatin (SS) have previously been shown to be reduced in the cortex and hippocampus of demented parkinsonian patients and patients with Alzheimer's disease. In situ hybridisation histochemistry (ISHH) was performed with an 35S tail-labelled oligonucleotide DNA probe to human SS mRNA, to examine its expression within the striatum, medial medullary lamina (MML) and reticular thalamic nucleus in Parkinson's disease (PD) and in matched controls. A chronic unilaterally MPTP-lesioned L-DOPA-naive primate model was also examined for comparison of SS mRNA expression with that in human L-DOPA treated PD subjects. Quantitation of SS mRNA expression on emulsion dipped sections revealed a significant increase (82%) in the MML of the globus pallidus in PD (56.5 microm2 of silver grain/cell, n = 9 cases) compared to controls (26.3 microm2/cell, n = 13 cases, p < 0.01, Student's t-test), paralleling the increase previously observed by this group for NOS mRNA. SS mRNA expression was higher in the dorsolateral than ventromedial putamen in controls (p < 0.001; DL: 24.89 +/- SEM 1.35; VM: 17.96 +/- SEM 2.63; n = 14) but this gradient was lost in PD cases (p > 0.05; DL: 22.68 +/- 1.94; VM: 22.17 +/- 2.94; n = 10). These findings suggest specific modification of basal ganglia SS-ergic pathways in PD.

A simplified and rapid procedure for in situ hybridization on human, flash-frozen, post-mortem brain and its combination with immunohistochemistry.

A simplified and rapid method is described for in situ hybridization (ISHH) studies of human post-mortem brain. Brain tissue was dissected into slices and was flash-frozen at -70 degrees C for storage. ISHH was carried out on 12 microns cryostat sections, post-fixed in 4% paraformaldehyde. The histology of human brain tissue prepared by this technique rivalled that of formalin-fixed, wax-embedded tissue. In ISHH studies, flash-frozen tissue gave superior results to those obtained following long-term fixation of tissue in 10% formalin with subsequent wax-embedding, or short-term prefixation in 4% paraformaldehyde. A systematic evaluation of commonly employed preparative procedures for ISHH was carried out on flash-frozen brain and a simplified protocol, consisting only of fixation and dehydration, was developed as a result of these studies. Specific hybridization of probes to a number of mRNA species was demonstrable in neurons in different brain regions. Using 0.5% glutaraldehyde/4% paraformaldehyde post-fixation, immunohistochemical labelling of TH-positive cortical catecholaminergic neurons and striatal dopaminergic terminals was successfully demonstrated in flash-frozen tissue. The same fixation technique also allowed combination of ISHH and immunohistochemistry for the simultaneous demonstration of tyrosine hydroxylase mRNA and peptide in neurons of human brain stem and cortex. mRNA and peptides in flash-frozen tissue were found to be stable for more than 3 years. ISHH could be readily performed on relatively large brain structures. In addition to permitting excellent ISHH and immunohistochemistry, alone or in combination, flash-freezing allows the maximum versatility of tissue use and does not compromise its study by other neuroscience techniques.

Glutamate decarboxylase-67 messenger RNA expression in normal human basal ganglia and in Parkinson's disease.

Expression of glutamate decarboxylase-67 messenger RNA was examined in the basal ganglia of normal controls and of cases of Parkinson's disease using in situ hybridization histochemistry in human post mortem material. In controls glutamate decarboxylase-67 messenger RNA expression was detected in all large neurons in both segments of the globus pallidus and in three neuronal subpopulations in the striatum as well as in substantia nigra reticulata neurons and in a small sub-population of subthalamic neurons. In Parkinson's disease, there was a statistically significant decrease of 50.7% in glutamate decarboxylase-67 messenger RNA expression per neuron in the lateral segment of the globus pallidus (controls: mean 72.8 microns2 +/- S.E.M. 8.7 of silver grain/neuron, n = 12; Parkinson's disease: mean 35.9 microns2 +/- S.E.M. 9.7 of silver grain/neuron, n = 9, P = 0.01, Student's t-test). In the medial segment of the globus pallidus, there was a small, but non-significant decrease of glutamate decarboxylase-67 messenger RNA expression in Parkinson's disease (controls: mean 100.6 microns2 +/- S.E.M. 7.2 of silver grain/neuron, n = 11; Parkinson's disease: mean 84.8 microns2 +/- S.E.M. 13.0 of silver grain/neuron, n = 7, P = 0.1, Student's t-test). No significant differences in glutamate decarboxylase-67 messenger RNA were detected in striatal neuronal sub-populations between Parkinson's disease cases and controls. These results are the first direct evidence in humans that there is increased inhibitory drive to the lateral segment of the globus pallidus in Parkinson's disease, as suggested by data from animal models. We therefore provide theoretical support for current experimental neurosurgical approaches to Parkinson's disease.

Dopamine transporter (Dat) and synaptic vesicle amine transporter (VMAT2) gene expression in the substantia nigra of control and Parkinson's disease.

The cellular expression of DAT mRNA and VMAT2 mRNA was investigated in sections of the human post-mortem substantia nigra in control and Parkinson's disease tissue using in situ hybridisation techniques. Short synthetic oligodeoxynucleotides were used to detect these gene transcripts at the cellular level. In the control human nigra, high levels of expression were seen in all sub-divisions of the substantia nigra, especially within medial regions. By contrast, the level of expression of both DAT mRNA and VMAT2 mRNA was markedly reduced in Parkinson's disease; these reductions in hybridisation signal were associated with (i) a marked loss of dopamine-containing cells in the substantia nigra, and (ii) a reduction in both DAT and VMAT2 signal per cell in the remaining pigmented neurones. These disease-related decreases in the cellular abundance of both DAT and VMAT2 gene transcripts in the surviving cells of the parkinsonian nigra may reflect compensatory changes in catecholamine signalling or may be a consequence of neuronal dysfunction.

Tissue pH as an indicator of mRNA preservation in human post-mortem brain.

The relationship between pH and mRNA preservation in post-mortem human brain was examined using in situ hybridization histochemistry and Northern hybridization with oligonucleotide probes in a large group of human subjects, including control and neuropathological cases. Tissue pH was found to correlate strongly with preservation of four mRNA species in three brain areas. Tissue with low pH, assumed to result from prolonged terminal hypoxia, contained reduced or absent mRNA, while tissue with higher pH was found to contain quantifiable amounts, the values for pathological brain samples being comparable to those for control material of similar pH. Measurement of tissue pH provides a simple means to screen post-mortem brain for mRNA preservation and is suggested as a means to match material in case-control studies of human neurodegenerative disease.

Does thyroid hormone influence the maturation of cerebellar granule neurones?

The following hypotheses were tested: is the degeneration of differentiating granule cells in the internal granular layer of the thyroid deficient cerebellum due to a direct requirement of these cells for thyroid hormone, or is it mediated through the failure of some of these cells to make synaptic contact with the hypoplastic Purkinje cells? The effect of thyroid hormone (T3) was studied in rat cerebellar cultures which contain predominantly granule cells. The cultures were grown in a chemically defined medium (S-) in the presence or absence of T3, and were also compared with serum (and thus thyroid hormone) containing cultures (S+). It would appear that T3 is not essential for the relatively long-term survival of the granule cells. Furthermore, cell growth in terms of protein accretion, and the morphological appearance of the cultures were also similar in S- in the presence and absence of T3. Maturation of granule cells was followed by estimating indices, which in the cerebellum in vivo are influenced by the hormone. However, developmental changes affecting the D2 protein, which is implicated in adhesion among nerve cells, and muscarinic receptor binding were not influenced by T3 in vitro. The voltage (veratridine)-sensitive uptake of 22Na was also unaffected, although T3 increased the rate of the relatively small veratridine insensitive component of the 22Na-influx. However, in comparison with cells grown in S+, the rate of both the veratridine sensitive and insensitive component of 22Na-influx was similar under serum-free conditions, whereas the maturation of the D2 protein and muscarinic receptor binding was retarded. The failure of thyroid hormone to influence the differentiation of granule cells, is not due to the in vitro conditions, since T3 is known to have a marked effect on the maturation of certain other classes of neural cells in culture. The results are consistent with the view that the effect of thyroid hormone on neural maturation is cell-type specific, and that granule cells are not targets of thyroid hormone action. They support our second hypothesis, that is, that the degeneration of granule cells in thyroid deficiency is a consequence of the reduction in available postsynaptic sites for the granule cell axons due to the retarded differentiation of Purkinje cells.

Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium.

Cultures obtained from early postnatal rat cerebellum, grown in either chemically defined or in serum-supplemented medium containing 25 mM K+, contained predominantly (greater than 90%) small interneurones, mostly granule cells, with good and comparable viability (assessed by the retention of preloaded 51Cr). Neuronal survival was prolonged in the chemically defined medium, nerve cells living up to two weeks longer than in serum-supplemented medium, although the proportion of non-neuronal cells was not greatly increased. In the serum-supplemented medium neurones became organised into clumps connected by thick, fasciculated bundles of neurites by about one week in vitro. In comparison, in the chemically defined medium aggregation of neurones and fasciculation of neurites was markedly reduced even after 4 weeks in culture. The possible relationship between the organisation of neurones and the nature of the substratum, chemical factors in the medium as well as the surface properties of the cells is discussed.

Localization of high affinity [3H]glycine transport sites in the cerebellar cortex.

A study was made of [3H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [3H]glycine at the low concentration used. Glial cells also took up [3H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [3H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [3H]GABA uptake (Ki over 600 microM vs the Kt of about 20 microM) and that GABA is an even weaker inhibitor of [3H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [3H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [3H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.

Effects of thyroid state on brain development: beta-adrenergic receptors and 5'-nucleotidase activity.

The effect of thyroid status on beta-adrenergic receptor binding and 5'-nucleotidase activity was studied in the forebrain and the cerebellum of the rat during the first 5 postnatal weeks. The developmental increase in beta-adrenergic receptor binding was significantly depressed in thyroid deficiency in both the forebrain and the cerebellum. The effect was more pronounced in the cerebellum, where at day 35 the concentration and the total number of beta-adrenergic receptor sites were reduced by 35% and 50% respectively. In contrast, hyperthyroidism had no significant effect on the development of beta-adrenergic receptors in the brain. On the other hand, hyperthyroidism led to a sustained increase in the forebrain in the activity of 5'-nucleotidase, an enzyme which is also associated with plasma membranes and has been proposed to play some role in neurotransmission. In thyroid deficiency the enzyme activity was markedly depressed. The effect was significant from day 12 in the cerebellum and from day 21 in the forebrain, the maximal depression, at day 21, being 55% and 45% respectively. In comparison with these plasma membrane markers, the accretion of membranous proteins was less affected: although this was retarded in hypothyroidism and advanced in hyperthyroidism there was no residual effect at 35 days except those attributable to changes in organ size. The results indicated, therefore, that the biochemical specialization of cells, as reflected in certain plasma membrane constituents, are chatacteristically influenced in the developing brain by thyroid disorders.