Empirically derived formulae for calculation of age- and region-related levels of iron, copper and zinc in the adult C57BL/6 mouse brain.

Metal dyshomeostasis is associated with neurodegenerative disorders, cancers and vascular disease. We report the effects of age (range: 3 to 18 months) on regional copper, iron and zinc levels in the brain of the C57BL/6 mouse, a widely used inbred strain with a permissive background allowing maximal expression of mutations in models that recapitulate these disorders. We present formulae that can be used to determine regional brain metal concentrations in the C57BL/6 mouse at any age in the range of three to eighteen months of life. Copper levels in the C57BL/6 mouse adult brain were highest in the striatum and cerebellum and increased with age, excepting the cortex and hippocampus. Regional iron levels increased linearly with age in all brain regions, while regional zinc concentrations became more homogeneous with age. Knockdown of the copper transporter Ctr1 reduced brain copper, but not iron or zinc, concentrations in a regionally-dependent manner. These findings demonstrate biometals in the brain change with age in a regionally-dependent manner. These data and associated formulae have implications for improving design and interpretation of a wide variety of studies in the C57BL/6 mouse.

Putative presynaptic dopamine dysregulation in schizophrenia is supported by molecular evidence from post-mortem human midbrain.

The dopamine hypothesis of schizophrenia posits that increased subcortical dopamine underpins psychosis. In vivo imaging studies indicate an increased presynaptic dopamine synthesis capacity in striatal terminals and cell bodies in the midbrain in schizophrenia; however, measures of the dopamine-synthesising enzyme, tyrosine hydroxylase (TH), have not identified consistent changes. We hypothesise that dopamine dysregulation in schizophrenia could result from changes in expression of dopamine synthesis enzymes, receptors, transporters or catabolic enzymes. Gene expression of 12 dopamine-related molecules was examined in post-mortem midbrain (28 antipsychotic-treated schizophrenia cases/29 controls) using quantitative PCR. TH and the synaptic dopamine transporter (DAT) proteins were examined in post-mortem midbrain (26 antipsychotic-treated schizophrenia cases per 27 controls) using immunoblotting. TH and aromatic acid decarboxylase (AADC) mRNA and TH protein were unchanged in the midbrain in schizophrenia compared with controls. Dopamine receptor D2 short, vesicular monoamine transporter (VMAT2) and DAT mRNAs were significantly decreased in schizophrenia, with no change in DRD3 mRNA, DRD3nf mRNA and DAT protein between diagnostic groups. However, DAT protein was significantly increased in putatively treatment-resistant cases of schizophrenia compared to putatively treatment-responsive cases. Midbrain monoamine oxidase A (MAOA) mRNA was increased, whereas MAOB and catechol-O-methyl transferase mRNAs were unchanged in schizophrenia. We conclude that, whereas some mRNA changes are consistent with increased dopamine action (decreased DAT mRNA), others suggest reduced dopamine action (increased MAOA mRNA) in the midbrain in schizophrenia. Here, we identify a molecular signature of dopamine dysregulation in the midbrain in schizophrenia that mainly includes gene expression changes of molecules involved in dopamine synthesis and in regulating the time course of dopamine action.

Selective cell death in neurodegeneration: why are some neurons spared in vulnerable regions?

Neurodegenerative diseases exhibit varying and characteristic patterns of regional brain cell death, yet in each disorder there are distinct variations in the relative vulnerability of neurons within targeted brain regions. For example, in Parkinson's disease (PD) up to 90% of dopaminergic neurons of the ventral tier of the substantia nigra pars compacta are lost at post-mortem, while as few as 25% of dopaminergic neurons in the dorsal tier of this nucleus succumb to the disease process. To date the reasons underlying differential vulnerability of similar neurons within a defined cytoarchitectural region has received little attention. We suggest variations in protein distribution underlies this differential vulnerability. Within the substantia nigra pars compacta the more vulnerable neurons exhibit an increased expression of factors that may contribute to vulnerability (D(2) dopamine autoreceptors, GIRK-2 potassium channels, lactotransferrin and the dopamine transporter) while also expressing a relative lack of neuroprotective elements (dopamine vesicle transport protein and a number of trophic and growth factors). Differential distribution of key proteins expressed by individual cells within the same cytoarchitectural brain region may influence the severity and likelihood of common neurodegenerative mechanisms, such as protein aggregation, oxidative stress, neuroinflammation and apoptosis, and thus the eventual fate of individual cells in the disease process. An understanding of how variable protein expression can influence cell survival within the diseased human brain in a range of neurodegenerative disorders may provide avenues for the development of novel strategies to improve the survival of targeted neurons in vivo.

Substantia nigra echomorphology and motor cortex excitability.

The aim of our study was to investigate the relation between substantia nigra (SN) echomorphology and indices of motor cortex excitability. Nigral hyperechogenicity in healthy individuals is thought to represent an SN abnormality or predisposition to Parkinson's disease (PD) and its prevalence is greater in the very old. Our study involved 20 old healthy subjects (aged 72-84 years) known to have normal (n=10) or abnormal (n=10) SN echomorphology. All were in good health with no overt neurological signs. SN morphology was assessed with transcranial sonography through the pre-auricular bone window. Motor cortical excitability and intracortical inhibition were assessed with transcranial magnetic stimulation (TMS) over the first dorsal interosseus motor area. Single stimuli were delivered during relaxation and voluntary contraction and paired stimuli were delivered during relaxation. Each cortical hemisphere was analysed separately. The response to single-pulse TMS (in motor cortex ipsilateral to the target SN) did not differ between groups. However, a significant difference between groups was observed in the paired pulse paradigm (conditioning stimulus intensity: 70% resting motor threshold; interstimulus interval: 2 ms). The conditioned motor evoked potential amplitude was significantly larger ipsilateral to the hyperechogenic SN than in controls (P=0.014). Thus, healthy subjects with SN hyperechogenicity exhibit significantly less intracortical inhibition within the motor cortex than subjects with normal echomorphology. Decreased intracortical inhibition is also observed in PD patients. This study provides further evidence that SN hyperechogenicity in healthy individuals is associated with changes characteristic of PD supporting a role for this feature as a vulnerability marker or state marker for subtle nigral dopaminergic dysfunction.

Anti-melanin antibodies are increased in sera in Parkinson's disease.

An increasing body of research suggests that a number of immune mechanisms play a role in degenerative pathways in Parkinson's disease (PD). In the current work we investigated a posited humoral immune response in this disorder. Sera from PD patients exhibited a significantly enhanced absorbance response on a novel ELISA for anti-melanin antibodies, compared to sera from age-matched control subjects. The enhanced ELISA absorbance response was specific for catecholamine-based melanins and was unrelated to antiparkinsonian dopaminergic medication. Further, the absorbance response was significantly and negatively correlated with disease duration. These data suggest that a specific humoral anti-melanin antibody response is present in PD and is more active in early disease. While the contribution of this novel immune response to the initiation and progression of this disorder is unclear, this finding supports the hypothesis that specific immune responses occurring in PD may respond to therapeutic interventions in this disorder.

White matter loss in healthy ageing: a postmortem analysis.

Age-related brain changes are widely documented. Because of differences in measurement methods and case selection, the reported effects of age on regional grey and white matter brain volumes, however, are much more pronounced and widespread in neuroimaging than in postmortem studies. Consequently, the magnitude of the effect that is specific to chronological age remains unresolved. We present postmortem volume measurements for 26 cortical, subcortical and white matter regions, in 24 human brains aged 46-92 years, free of neuropathological abnormalities. Significant age-related loss was observed in anterior and posterior white matter but not in total grey matter volumes. Further analyses on five cortical subregions previously reported to exhibit large age-related loss on MRI yielded negative results. These analyses demonstrate smaller changes with age than those reported in imaging studies. Although this discrepancy between postmortem and imaging studies may partly be explained by the increase in noise of the neuroimaging data with age, our results suggest that healthy brain ageing is a process affecting predominantly white matter not grey matter.

The comparative biology of neuromelanin and lipofuscin in the human brain.

Neuromelanin and lipofuscin are two pigments produced within the human brain that, until recently, were considered inert cellular waste products of little interest to neuroscience. Recent research has increased our understanding of the nature and interactions of these pigments with their cellular environment and suggests that these pigments may, indeed, influence cellular function. The physical appearance and distribution of the pigments within the human brain differ, but both accumulate in the aging brain and the pigments share some structural features. Lipofuscin accumulation has been implicated in postmitotic cell aging, while neuromelanin is suggested to function as an iron-regulatory molecule with possible protective functions within the cells which produce this pigment. This review presents comparative aspects of the biology of neuromelanin and lipofuscin, as well as a discussion of their hypothesized functions in brain and their possible roles in aging and neurodegenerative disease.

Lipid content determines aggregation of neuromelanin granules in vitro.

The neuromelanin pigment of the substantia nigra of the human brain is closely associated with lipids and other non-melanogenic compounds which appear to contribute to the unique and complex morphology of neuromelanin pigment granules. In this work we show that insoluble granules isolated from the human substantia nigra associate in vitro to form pigment aggregates similar to those present in the human brain. Extraction of neuromelanin-associated polar lipids by methanol and/or hexane significantly enhanced melanin aggregate size. A marked (10-fold) increase in granule size was seen after methanol treatment, whereas the application of hexane after methanol reduced this pro-aggregation effect. We have previously reported that hexane and methanol remove the neuromelanin-associated polyisoprenoids dolichol and cholesterol respectively. Thus, the current data suggests that pigment-associated lipids may be a factor regulating pigment aggregation and neuromelanin granule size in vivo.

Substantia nigra echomorphology in the healthy very old: Correlation with motor slowing.

Transcranial sonography reveals an increase in echogenicity in the substantia nigra of patients with idiopathic Parkinson's disease. Marked hyperechogenicity has also been described in 9% of the healthy population and is associated with subtle clinical or functional neuroimaging findings suggestive of changes in nigrostriatal function. It has therefore been hypothesised that a hyperechogenic substantia nigra represents an early stage of nigral degeneration or a predisposition for Parkinson's disease. In the present study, we correlated sonographic findings with motor and cognitive deficits in a group of healthy, very elderly subjects. Marked and moderately increased substantia nigra echogenicity was present in 25% and 21% of our healthy, very elderly subjects, respectively, and correlated strongly with the presence of extrapyramidal symptoms in the absence of cognitive deficits. The high incidence of substantia nigra hyperechogenicity measured in our very elderly subjects compared with previous TCS studies suggests that the prevalence of this feature increases with age and is consistent with the higher prevalence of Parkinson's disease in advanced age, as well as the increased frequency of extrapyramidal symptoms. Our results indicate that this simple technique can be used to identify and quantify brain changes associated with subtle motor dysfunction in the very elderly.

New face of neuromelanin.

The massive, early and relatively circumscribed death of the dopaminergic neurons of the substantia nigra in Parkinson's disease has not yet been adequately explained. The characteristic feature of this brain region is the presence of neuromelanin pigment within the vulnerable neurons. We suggest that neuromelanin in the Parkinson's disease brain differs to that in the normal brain. The interaction of neuromelanin with iron has been shown to differ in the parkinsonian brain in a manner consistent with an increase in oxidative stress. Further, we suggest an interaction between the lipoprotein alpha-synuclein and lipidated neuromelanin contributes to the aggregation of this protein and cell death in Parkinson's disease. The available data suggest that the melaninisation of the dopaminergic neurons of the substantia nigra is a critical factor to explain the vulnerability of this brain region to early and massive degeneration in Parkinson's disease.

Potential sources of increased iron in the substantia nigra of parkinsonian patients.

Histopathological, biochemical and in vivo brain imaging techniques, such as magnetic resonance imaging and transcranial sonography, revealed a consistent increase of substantia nigra (SN) iron in Parkinson's disease (PD). Increased iron deposits in the SN may have genetic and non-genetic causes. There are several rare movement disorders associated with neurodegeneration, and genetic abnormalities in iron regulation resulting in iron deposition in the brain. Non-genetic causes of increased SN iron may be the result of a disturbed or open blood-brain-barrier, local changes in the normal iron-regulatory systems, intraneuronal transportation of iron from iron-rich area into the SN and release of iron from intracellular iron storage molecules. Major iron stores are ferritin and haemosiderin in glial cells as well as neuromelanin in neurons. Age- and disease dependent overload of iron storage proteins may result in iron release upon reduction. Consequently, the low molecular weight chelatable iron complexes may trigger redox reactions leading to damage of biomolecules. Additionally, upon neurodegeneration there is strong microglial activation which can be another source of high iron concentrations in the brain.

Investigation of the lipid component of neuromelanin.

OBJECTIVE: Neuromelanin (NM) is different to other melanins in that its ultrastructure includes a lipid component. The objectives of this study were to identify and quantify lipids associated with NM. RESULTS: Quantification of the lipid component associated with the pigment on electron micrographs demonstrated that this component comprises 35% of the NM granule volume in the normal brain. The irregular ultrastructural appearance of the NM granules was quite different to the round regular boundary of melanin granules. Using reversed phase high performance liquid chromatography (HPLC) coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry we demonstrated that the isoprenoid dolichol accounted for approximately 12% of total NM pigment mass. Low levels of other lipids were detectable (cholesterol, ubiquinone-10 and alpha-tocopherol) and account for <0.05% of NM lipid, in contrast to cholesterol accounting for 35% of total brain lipids. CONCLUSION: Unlike other melanins, a substantial proportion of NM volume is comprised of lipid and the major type of lipid associated with NM granules is the isoprenoid dolichol.

Functional effects of neuromelanin and synthetic melanin in model systems.

The function of the dark polymer pigment neuromelanin found in catecholaminergic neurons of the human brain is not understood, especially as most published data are based upon a synthetic model melanin which differs structurally to the native pigment. Nevertheless human neuromelanin has been shown to efficiently bind transition metals such as iron, as well as other potentially toxic molecules. The pigment may have a protective function in the healthy brain by, for example, contributing to iron homeostasis within pigmented nuclei. We have demonstrated that synthetic dopamine melanin stimulates cell damage in both cell lines and primary cells in vitro, an effect associated with increased hydroxyl radical production and apoptosis. In contrast, at low iron concentrations native neuromelanin does not induce cell damage but rather protects cells in culture from oxidative stress. This protective function appears to be lost at high iron concentrations where neuromelanin saturated with iron functions as a source of oxidative load, rather than an iron chelator. Changes to neuromelanin and tissue iron load in Parkinson's disease may decrease the protective potential of the pigment, thus increase the potential for cell damage in this disorder.

Evidence for specific phases in the development of human neuromelanin.

Neuromelanin is a dark-coloured pigment which forms in the dopamine neurons of the human midbrain. Here we describe the age-related development and regulation of neuromelanin within these dopamine neurons. 10 microm sections from formalin-fixed midbrain from 29 people spanning the ages of 24 weeks to 95 years old were either stained with a basic Nissl substance stain (0.5% cresyl violet), or processed unstained. After locating the substantia nigra using the stained sections, digital photos were taken of individual ventral substantia nigra neurons in the unstained sections, and the cellular area occupied by pigment, and optical density were measured using computer software. These measurements demonstrated three developmental phases. Neuromelanin was not present at birth and initiation of pigmentation began at approximately 3 years of age, followed by a period of increasing pigment granule number and increasing pigment granule colouration until age 20. In middle and later life the colour of the pigment granules continued to darken but was not associated with any substantial growth in pigment volume. The identification of three phases and changes in the rate of neuromelanin production over time suggests the regulation of neuromelanin production and turnover, possibly through enzymatic processes.

Evidence for specific phases in the development of human neuromelanin.

Neuromelanin is a dark-coloured pigment which forms in the dopamine neurons of the human midbrain. The age-related development and regulation of neuromelanin within these dopamine neurons has not been previously described. Optical density and area measurements of unstained neuromelanin in ventral substantia nigra neurons from 29 people spanning the ages of 24 weeks to 95 years old, demonstrated three developmental phases. Neuromelanin was not present at birth and initiation of pigmentation began at approximately 3 years of age, followed by a period of increasing pigment granule number and increasing pigment granule colouration until age 20. In middle and later life the colour of the pigment granules continued to darken but was not associated with any substantial growth in pigment volume. The identification of three phases and changes in the rate of neuromelanin production over time suggests the regulation of neuromelanin production and turnover, possibly through enzymatic processes.

Neuromelanin in human dopamine neurons: comparison with peripheral melanins and relevance to Parkinson's disease.

Neuromelanin (NM) is a dark polymer pigment produced in specific populations of catecholaminergic neurons in the brain. It appears in greatest quantities in the human brain, in lesser amounts in some other non-human primates, but is absent from the brain in many lower species. Interest in this pigment has seen a resurgence in recent years because of a hypothesised link between neuromelanin and the especial vulnerability of neuromelanin-containing neurons to cell death in Parkinson's disease (PD). Little is known regarding the biology of neuromelanin. As neuromelanin appears to have characteristics in common with the better studied peripheral melanin pigments this review compares what is known about neuromelanin with melanins found in other body tissues. Unlike peripheral melanins, which are produced in specialised cells called melanocytes and may be transferred to other cell types, neuromelanin granules are believed to be stored in the cell in which they are produced. Neuromelanin granules display a unique, more heterogeneous appearance compared with peripheral melanins. Unlike melanin, neuromelanin is traditionally thought to result from a non-enzymatic synthesis pathway with no known pathway for neuromelanin catabolism. More recent data, however, is indicative of some regulation of neuromelanin synthesis and turnover. By analogy with peripheral melanins, neuromelanin may function in vivo to attenuate the effects of damaging stimuli. Among several possible mechanisms suggested, the ability of neuromelanin to interact with transition metals, especially iron, and to mediate intracellular oxidative mechanisms has received particular attention. Recent data from neuromelanin in the Parkinson's disease brain suggests that this proposed function may be compromised, thus rendering pigmented neurons vulnerable to oxidative damage in this disorder.

Dopamine receptor agonists in the therapy of Parkinson's disease.

Forty years after its introduction by Birkmayer and Hornykiewicz (1961), L-DOPA-based therapy of Parkinson's disease remains the central pillar in the management of the disorder. Nevertheless, it is not unproblematic, and dopamine receptor agonists play increasingly important roles in antiparkinsonian therapy. Pharmacological and pharmacokinetic properties of these agents are briefly reviewed and followed by a detailed summary of available literature concerning controlled trials in Parkinson's disease. It is concluded that there is little unequivocal evidence to suggest that any of the major dopamine receptor agonists should be invariably preferred in the therapy of Parkinson's disease; their application must be based on the needs and responses of individual patients.

The dopamine receptor agonist lisuride attenuates iron-mediated dopaminergic neurodegeneration.

Many dopamine agonists used in the treatment of Parkinson's disease are suggested to be potentially neuroprotective. On the basis of its structure, the dopamine agonist lisuride may share this characteristic. In the current study discrete asymptomatic lesions were produced by the injection of iron-laden neuromelanin into the rat substantia nigra and the animals treated with lisuride to determine the protective potential of this substance. Two treatment regimes were utilised. In the neuroprotective protocol, animals were treated with 0.1 mg.kg(-1) lisuride twice daily 3 days prior to, and 7 days following, the iron lesion. In the neurorescue protocol, the animals received 0.1 mg.kg(-1) lisuride twice daily for 1 week beginning on the fourth day post surgery. Eight weeks post surgery, tyrosine hydroxylase-positive neurons surrounding the injection site (33% of total nigral volume) were counted. Dopamine neuron number in iron-lesioned animals was reduced to 50% of that in vehicle-injected animals. The absence of motoric disturbances or a striatal dopamine deficit in these animals suggests a subclinical dopaminergic lesion. Dopamine neuron number in the quantified area in sham-injected animals receiving lisuride or iron-lesioned animals receiving lisuride in both the neuroprotection and neurorescue groups were not significantly reduced. These results suggest that lisuride can protect neurons against iron-induced cell death and might thus be neuroprotective in Parkinson's disease.

Identifying severely atrophic cortical subregions in Alzheimer's disease.

The present study analyses the pattern of atrophy in anatomically discrete brain regions in prospectively-studied pathologically-confirmed patients with Alzheimer's disease (AD) and controls. Standard volumetric measurements were made of the entire cortex subdivided into 23 anatomical regions. Analyses determined regions of significant atrophy in AD and differences between the severity and rates of atrophy. Two levels of severity were found. Atrophy concentrated in medial temporal lobe structures as well as in inferior temporal and superior and middle frontal cortices. The degree of atrophy in these regions related to disease duration, consistent with an early and sustained disease process. The rate of atrophy was significantly greater for the fusiform gyrus. The inferior frontal lobes were entirely spared at all AD stages, while atrophy of other cortical regions was less marked and not related to disease duration, suggesting late involvement. Our findings show that the fusiform gyrus is particularly affected by AD, and suggest two levels of atrophy that correspond with published neurofibrillary tangle (most atrophic) and senile plaque (less atrophic) densities.

Influence of neuromelanin on oxidative pathways within the human substantia nigra.

Neuromelanin (NM) is a dark-coloured pigment produced in the dopaminergic neurons of the human substantia nigra (SN). The function of NM within the pigmented neurons is unknown but other melanins are believed to play a protective role via attenuation of free radical damage. Experimental evidence suggests that NM may also exhibit this characteristic, possibly by directly inactivating free radical species or via its ability to chelate transition metals, such as iron. Increased tissue iron, however, may saturate iron-chelating sites on NM and a looser association between iron and NM may result in an increased, rather than decreased, production of free radical species. The death of NM-pigmented neurons in Parkinson's disease (PD) is associated with both a measurable increase in tissue iron concentrations and indices of free radical mediated damage, suggesting that NM is involved in the aetiology of this disorder. As yet, it is unknown whether NM in the parkinsonian brain differs to that found in healthy tissue and thus may fulfil a different role within this tissue.