A Novel Mitochondria-Targeting Iron Chelator Neuroprotects Multimodally via HIF-1 Modulation Against a Mitochondrial Toxin in a Dopaminergic Cell Model of Parkinson's Disease.

Coumarins are plant-derived polyphenolic compounds belonging to the benzopyrones family, possessing wide-ranging pharmaceutical applications including cytoprotection, which may translate into therapeutic potential for multiple diseases, including Parkinson's disease (PD). Here we demonstrate the neuroprotective potential of a new polyhydroxyl coumarin, N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetamide (CT51), against the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+). MPP+'s mechanism of toxicity relates to its ability to inhibit complex I of the mitochondrial electron transport chain (METC), leading to adenosine triphosphate (ATP) depletion, increased reactive oxygen species (ROS) production, and apoptotic cell death, hence mimicking PD-related neuropathology. Dopaminergic differentiated human neuroblastoma cells were briefly pretreated with CT51, followed by toxin exposure. CT51 significantly restored somatic cell viability and neurite processes; hence, the drug targets cell bodies and axons thereby preserving neural function and circuitry against PD-related damage. Moreover, MPP+ emulates the iron dyshomeostasis affecting dopaminergic neurons in PD-affected brains, whilst CT51 was previously revealed as an effective iron chelator that preferentially partitions to mitochondria. We extend these findings by characterising the drug's interactive effects at the METC level. CT51 did not improve mitochondrial coupling efficiency. However, voltammetric measurements and high-resolution respirometry analysis revealed that CT51 acts as an antioxidant agent. Also, the neuronal protection afforded by CT51 associated with downregulating MPP+-induced upregulated expression of hypoxia-inducible factor 1 alpha (HIF-1α), a protein which regulates iron homeostasis and protects against certain forms of oxidative stress after translocating to mitochondria. Our findings support the further development of CT51 as a dual functioning iron chelator and antioxidant antiparkinsonian agent.

Mitochondrial DNA population variation is not associated with Alzheimer's in the Japanese population: A consistent finding across global populations.

Several mitochondrial DNA (mtDNA) haplogroup association studies have suggested that common mtDNA variants are associated with multifactorial diseases, including Alzheimer's disease (AD). However, such studies have also produced conflicting results. A new mtDNA association model, the 'variant load model' (VLM), has been applied to multiple disease phenotypes. Application of the VLM in a 2017 study failed to find different variant loads in AD patients compared to controls, in two cohorts of European origin. The study also suggested a lower variant load in healthy elderly individuals, but could offer no replicate cohort to support this observation. Here, the VLM is applied to Japanese mtDNA sequences; in doing so, we explored the role of mtDNA variation in AD and ageing in a different global population. Consistent with the previous findings using the VLM in two populations of European origin, we found no evidence for an association between rarer, non-haplogroup associated variation and the development of AD. However, the result in the context of ageing that suggested those with fewer mildly deleterious mutations might undergo healthier ageing, was not replicated. In contrast to our previous study, our present results suggest that those living to advanced old age may harbour more mildly deleterious mtDNA variations. Importantly our analysis showed this finding is not primarily being driven by many rare population variants dispersed across the mtDNA, but by a few more frequent variants with high MutPred scores. It is suggested the variants in question do not exert a mildly deleterious effect in their most frequent haplogroup context.

Mitochondrial DNA variation in Parkinson's disease: Analysis of "out-of-place" population variants as a risk factor.

Mitochondrial DNA (mtDNA), a potential source of mitochondrial dysfunction, has been implicated in Parkinson's disease (PD). However, many previous studies investigating associations between mtDNA population variation and PD reported inconsistent or contradictory findings. Here, we investigated an alternative hypothesis to determine whether mtDNA variation could play a significant role in PD risk. Emerging evidence suggests that haplogroup-defining mtDNA variants may have pathogenic potential if they occur "out-of-place" on a different maternal lineage. We hypothesized that the mtDNA of PD cases would be enriched for out-of-place variation in genes encoding components of the oxidative phosphorylation complexes. We tested this hypothesis with a unique dataset comprising whole mitochondrial genomes of 70 African ancestry PD cases, two African ancestry control groups (n = 78 and n = 53) and a replication group of 281 European ancestry PD cases and 140 controls from the Parkinson's Progression Markers Initiative cohort. Significantly more African ancestry PD cases had out-of-place variants than controls from the second control group (P < 0.0125), although this association was not observed in the first control group nor the replication group. As the first mtDNA study to include African ancestry PD cases and to explore out-of-place variation in a PD context, we found evidence that such variation might be significant in this context, thereby warranting further replication in larger cohorts.

Synthesis, physicochemical characterization and neuroprotective evaluation of novel 1-hydroxypyrazin-2(1H)-one iron chelators in an in vitro cell model of Parkinson's disease.

Iron dysregulation, dopamine depletion, cellular oxidative stress and α-synuclein protein mis-folding are key neuronal pathological features seen in the progression of Parkinson's disease. Iron chelators endowed with one or more therapeutic modes of action have long been suggested as disease modifying therapies for its treatment. In this study, novel 1-hydroxypyrazin-2(1H)-one iron chelators were synthesized and their physicochemical properties, iron chelation abilities, antioxidant capacities and neuroprotective effects in a cell culture model of Parkinson's disease were evaluated. Physicochemical properties (log ß, log D7.4, pL0.5) suggest that these ligands have a poorer ability to penetrate cell membranes and form weaker iron complexes than the closely related 1-hydroxypyridin-2(1H)-ones. Despite this, we show that levels of neuroprotection provided by these ligands against the catecholaminergic neurotoxin 6-hydroxydopamine in vitro were comparable to those seen previously with the 1-hydroxypyridin-2(1H)-ones and the clinically used iron chelator Deferiprone, with two of the ligands restoring cell viability to ≥89% compared to controls. Two of the ligands were endowed with additional phenol moieties in an attempt to derive multifunctional chelators with dual iron chelation/antioxidant activity. However, levels of neuroprotection with these ligands were no greater than ligands lacking this moiety, suggesting the neuroprotective properties of these ligands are due primarily to chelation and passivation of intracellular labile iron, preventing the generation of free radicals and reactive oxygen species that otherwise lead to the neuronal cell death seen in Parkinson's disease.

Investigation of the correlation between mildly deleterious mtDNA Variations and the clinical progression of multiple sclerosis.

BACKGROUND: Evidence suggests that mitochondrial DNA (mtDNA) variation at a population level may influence susceptibility to, or the clinical progression of Multiple Sclerosis (MS). OBJECTIVE: To determine if mtDNA population variation is linked to the clinical progress of MS. METHODS: Using the complete mtDNA sequences of 217 MS patients, we applied the new 'variant load' model, designed as a framework by which to examine the role of mtDNA variation in the context of complex clinical disease. RESULTS: No significant association was detected between mtDNA 'variant load'and the clinical measures of progression. CONCLUSION: Our results suggest that mtDNA population variation does not play a substantial role in the clinical progression of MS; however, modest effects and/or effects in a subgroup of patients cannot be entirely excluded. Results do not exclude the possibility of detecting an association between variation and more strictly quantified variables obtained from histopathologically-stained specimens. The results further illustrate the method's applicabilityto other disease phenotypes.

Single-Cell Approaches for Studying the Role of Mitochondrial DNA in Neurodegenerative Disease.

In light of accumulating evidence suggestive of cell type-specific vulnerabilities as a result of normal aging processes that adversely affect the brain, as well as age-related neurodegenerative disorders such as Parkinson's disease (PD), the current chapter highlights how we study mitochondrial DNA (mtDNA) changes at a single-cell level. In particular, we comment on increasing questioning of the narrow neurocentric view of such pathologies, where microglia and astrocytes have traditionally been considered bystanders rather than players in related pathological processes. Here we review the contribution made by single-cell mtDNA alterations towards neuronal vulnerability seen in neurodegenerative disorders, focusing on PD as a prominent example. In addition, we give an overview of methodologies that support such experimental investigations. In considering the significant advances that have been made in recent times for developing mitochondria-specific therapies, investigations to account for cell type-specific mitochondrial patterns and how these are altered by disease hold promise for delivering more effective disease-modifying therapeutics.

Dissociable contributions of mediodorsal and anterior thalamic nuclei in visual attentional performance: A comparison using nicotinic and muscarinic cholinergic receptor antagonists.

BACKGROUND: Thalamic subregions mediate various cognitive functions, including attention, inhibitory response control and decision making. Such neuronal activity is modulated by cholinergic thalamic afferents and deterioration of such modulatory signaling has been theorised to contribute to cognitive decline in neurodegenerative disorders. However, the thalamic subnuclei and cholinergic receptors involved in cognitive functioning remain largely unknown. AIMS: We investigated whether muscarinic or nicotinic receptors in the mediodorsal thalamus and anterior thalamus contribute to rats' performance in the five-choice serial reaction time task, which measures sustained visual attention and impulsive action. METHODS: Male Long-Evans rats were trained in the five-choice serial reaction time task then surgically implanted with guide cannulae targeting either the mediodorsal thalamus or anterior thalamus. Reversible inactivation of either the mediodorsal thalamus or anterior thalamus were achieved with infusions of the γ-aminobutyric acid-ergic agonists muscimol and baclofen prior to behavioural assessment. To investigate cholinergic mechanisms, we also assessed the behavioural effects of locally administered nicotinic (mecamylamine) and muscarinic (scopolamine) receptor antagonists. RESULTS: Reversible inactivation of the mediodorsal thalamus severely impaired discriminative accuracy and response speed and increased omissions. Inactivation of the anterior thalamus produced less profound effects, with impaired accuracy at the highest dose. In contrast, blocking cholinergic transmission in these regions did not significantly affect five-choice serial reaction time task performance. CONCLUSIONS/INTERPRETATIONS: These findings show the mediodorsal thalamus plays a key role in visuospatial attentional performance that is independent of local cholinergic neurotransmission.

Design and evaluation of bi-functional iron chelators for protection of dopaminergic neurons from toxicants.

While the etiology of non-familial Parkinson's disease (PD) remains unclear, there is evidence that increased levels of tissue iron may be a contributing factor. Moreover, exposure to some environmental toxicants is considered an additional risk factor. Therefore, brain-targeted iron chelators are of interest as antidotes for poisoning with dopaminergic toxicants, and as potential treatment of PD. We, therefore, designed a series of small molecules with high affinity for ferric iron and containing structural elements to allow their transport to the brain via the neutral amino acid transporter, LAT1 (SLC7A5). Five candidate molecules were synthesized and initially characterized for protection from ferroptosis in human neurons. The promising hydroxypyridinone SK4 was characterized further. Selective iron chelation within the physiological range of pH values and uptake by LAT1 were confirmed. Concentrations of 10-20 µM blocked neurite loss and cell demise triggered by the parkinsonian neurotoxicants, methyl-phenyl-pyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) in human dopaminergic neuronal cultures (LUHMES cells). Rescue was also observed when chelators were given after the toxicant. SK4 derivatives that either lacked LAT1 affinity or had reduced iron chelation potency showed altered activity in our assay panel, as expected. Thus, an iron chelator was developed that revealed neuroprotective properties, as assessed in several models. The data strongly support the role of iron in dopaminergic neurotoxicity and suggests further exploration of the proposed design strategy for improving brain iron chelation.

Novel 1-hydroxypyridin-2-one metal chelators prevent and rescue ubiquitin proteasomal-related neuronal injury in an in vitro model of Parkinson's disease.

Ubiquitin proteasome system (UPS) impairment, excessive cellular oxidative stress, and iron dyshomeostasis are key to substantia nigra dopaminergic neuronal degeneration in Parkinson's disease (PD); however, a link between these features remains unconfirmed. Using the proteasome inhibitor lactacystin we confirm that nigral injury via UPS impairment disrupts iron homeostasis, in turn increasing oxidative stress and promoting protein aggregation. We demonstrate the neuroprotective potential of two novel 1-hydroxy-2(1H)-pyridinone (1,2-HOPO) iron chelators, compounds C6 and C9, against lactacystin-induced cell death. We demonstrate that this cellular preservation relates to the compounds' iron chelating capabilities and subsequent reduced capacity of iron to form reactive oxygen species (ROS), where we also show that the ligands act as antioxidant agents. Our results also demonstrate the ability of C6 and C9 to reduce intracellular lactacystin-induced α-synuclein burden. Stability constant measurements confirmed a high affinity of C6 and C9 for Fe3+ and display a 3:1 HOPO:Fe3+ complex formation at physiological pH. Reducing iron reactivity could prevent the demise of nigral dopaminergic neurons. We provide evidence that the lactacystin model presents with several neuropathological hallmarks of PD related to iron dyshomeostasis and that the novel chelating compounds C6 and C9 can protect against lactacystin-related neurotoxicity.

DREADD Activation of Pedunculopontine Cholinergic Neurons Reverses Motor Deficits and Restores Striatal Dopamine Signaling in Parkinsonian Rats.

The brainstem-based pedunculopontine nucleus (PPN) traditionally associates with motor function, but undergoes extensive degeneration during Parkinson's disease (PD), which correlates with axial motor deficits. PPN-deep brain stimulation (DBS) can alleviate certain symptoms, but its mechanism(s) of action remains unknown. We previously characterized rats hemi-intranigrally injected with the proteasomal inhibitor lactacystin, as an accurate preclinical model of PD. Here we used a combination of chemogenetics with positron emission tomography imaging for in vivo interrogation of discrete neural networks in this rat model of PD. Stimulation of excitatory designer receptors exclusively activated by designer drugs expressed within PPN cholinergic neurons activated residual nigrostriatal dopaminergic neurons to produce profound motor recovery, which correlated with striatal dopamine efflux as well as restored dopamine receptor 1- and dopamine receptor 2-based medium spiny neuron activity, as was ascertained with c-Fos-based immunohistochemistry and stereological cell counts. By revealing that the improved axial-related motor functions seen in PD patients receiving PPN-DBS may be due to stimulation of remaining PPN cholinergic neurons interacting with dopaminergic ones in both the substantia nigra pars compacta and the striatum, our data strongly favor the PPN cholinergic-midbrain dopaminergic connectome as mechanism for PPN-DBS's therapeutic effects. These findings have implications for refining PPN-DBS as a promising treatment modality available to PD patients.

Altered motor, anxiety-related and attentional task performance at baseline associate with multiple gene copies of the vesicular acetylcholine transporter and related protein overexpression in ChAT::Cre+ rats.

Transgenic rodents expressing Cre recombinase cell specifically are used for exploring mechanisms regulating behavior, including those mediated by cholinergic signaling. However, it was recently reported that transgenic mice overexpressing a bacterial artificial chromosome containing choline acetyltransferase (ChAT) gene, for synthesizing the neurotransmitter acetylcholine, present with multiple vesicular acetylcholine transporter (VAChT) gene copies, resulting in altered cholinergic tone and accompanying behavioral abnormalities. Since ChAT::Cre+ rats, used increasingly for understanding the biological basis of CNS disorders, utilize the mouse ChAT promotor to control Cre recombinase expression, we assessed for similar genotypical and phenotypical differences in such rats compared to wild-type siblings. The rats were assessed for mouse VAChT copy number, VAChT protein expression levels and for sustained attention, response control and anxiety. Rats were also subjected to a contextual fear conditioning paradigm using an unconditional fear-inducing stimulus (electrical foot shocks), with blood samples taken at baseline, the fear acquisition phase and retention testing, for measuring blood plasma markers of hypothalamic-pituitary-adrenal gland (HPA)-axis activity. ChAT::Cre+ rats expressed multiple mouse VAChT gene copies, resulting in significantly higher VAChT protein expression, revealed anxiolytic behavior, hyperlocomotion and deficits in tasks requiring sustained attention. The HPA-axis was intact, with unaltered circulatory levels of acute stress-induced corticosterone, leptin and glucose. Our findings, therefore, reveal that in ChAT::Cre+ rats, VAChT overexpression associates with significant alterations of certain cognitive, motor and affective functions. Although highly useful as an experimental tool, it is essential to consider the potential effects of altered cholinergic transmission on baseline behavior in ChAT::Cre rats.

Regional mitochondrial DNA and cell-type changes in post-mortem brains of non-diabetic Alzheimer's disease are not present in diabetic Alzheimer's disease.

Diabetes increases the risk of Alzheimer's disease (AD), and mitochondrial dysfunction is implicated in both diseases, however the impact of both diabetes and AD on brain mitochondria is not known. We measured mitochondrial DNA (mtDNA), an indicator of mitochondrial function, in frontal, parietal, and cerebellar regions of post-mortem human brains (n = 74) from non-cognitively impaired controls (NCI), mild-cognitively impaired (MCI) and AD cases. In a subset of parietal cortices, we measured mRNAs corresponding to cell types and mitochondrial function and semi-automated stereological assessment was performed on immune-staining of parietal cortex sections. mtDNA showed significant regional variation, highest in parietal cortex, and lowest in cerebellum. Irrespective of cognitive status, all brain regions had significantly higher mtDNA in diabetic cases. In the absence of diabetes, AD parietal cortices had decreased mtDNA, reduced MAP2 (neuronal) and increased GFAP (astrocyte) mRNA, relative to NCI. However, in the presence of diabetes, we did not observe these AD-related changes, suggesting that the pathology observed in diabetic AD may be different to that seen in non-diabetic AD. The lack of clear functional changes in mitochondrial parameters in diabetic AD suggest different cellular mechanisms contributing to cognitive impairment in diabetes which remain to be fully understood.

The unresolved role of mitochondrial DNA in Parkinson's disease: An overview of published studies, their limitations, and future prospects.

Parkinson's disease (PD), a progressive neurodegenerative disorder, has long been associated with mitochondrial dysfunction in both sporadic and familial forms of the disease. Mitochondria are crucial for maintaining cellular homeostasis, and their dysfunction is detrimental to dopaminergic neurons. These neurons are highly dependent on mitochondrial adenosine triphosphate (ATP) and degenerate in PD. Mitochondria contain their own genomes (mtDNA). The role of mtDNA has been investigated in PD on the premise that it encodes vital components of the ATP-generating oxidative phosphorylation (OXPHOS) complexes and accumulates somatic variation with age. However, the association between mtDNA variation and PD remains controversial. Herein, we provide an overview of previously published studies on the role of inherited as well as somatic (acquired) mtDNA changes in PD including point mutations, deletions and depletion. We outline limitations of previous investigations and the difficulties associated with studying mtDNA, which have left its role unresolved in the context of PD. Lastly, we highlight the potential for further research in this field and provide suggestions for future studies. Overall, the mitochondrial genome is indispensable for proper cellular function and its contribution to PD requires further, more extensive investigation.

MtDNA population variation in Myalgic encephalomyelitis/Chronic fatigue syndrome in two populations: a study of mildly deleterious variants.

Myalgic Encephalomyelitis (ME), also known as Chronic Fatigue Syndrome (CFS) is a debilitating condition. There is growing interest in a possible etiologic or pathogenic role of mitochondrial dysfunction and mitochondrial DNA (mtDNA) variation in ME/CFS. Supporting such a link, fatigue is common and often severe in patients with mitochondrial disease. We investigate the role of mtDNA variation in ME/CFS. No proven pathogenic mtDNA mutations were found. We then investigated population variation. Two cohorts were analysed, one from the UK (n = 89 moderately affected; 29 severely affected) and the other from South Africa (n = 143 moderately affected). For both cohorts, ME/CFS patients had an excess of individuals without a mildly deleterious population variant. The differences in population variation might reflect a mechanism important to the pathophysiology of ME/CFS.

Understanding Miro GTPases: Implications in the Treatment of Neurodegenerative Disorders.

The Miro GTPases represent an unusual subgroup of the Ras superfamily and have recently emerged as important mediators of mitochondrial dynamics and for maintaining neuronal health. It is now well-established that these enzymes act as essential components of a Ca2+-sensitive motor complex, facilitating the transport of mitochondria along microtubules in several cell types, including dopaminergic neurons. The Miros appear to be critical for both anterograde and retrograde mitochondrial transport in axons and dendrites, both of which are considered essential for neuronal health. Furthermore, the Miros may be significantly involved in the development of several serious pathological processes, including the development of neurodegenerative and psychiatric disorders. In this review, we discuss the molecular structure and known mitochondrial functions of the Miro GTPases in humans and other organisms, in the context of neurodegenerative disease. Finally, we consider the potential human Miros hold as novel therapeutic targets for the treatment of such disease.

Temporal-Spatial Profiling of Pedunculopontine Galanin-Cholinergic Neurons in the Lactacystin Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is conventionally seen as resulting from single-system neurodegeneration affecting nigrostriatal dopaminergic neurons. However, accumulating evidence indicates multi-system degeneration and neurotransmitter deficiencies, including cholinergic neurons which degenerate in a brainstem nucleus, the pedunculopontine nucleus (PPN), resulting in motor and cognitive impairments. The neuropeptide galanin can inhibit cholinergic transmission, while being upregulated in degenerating brain regions associated with cognitive decline. Here we determined the temporal-spatial profile of progressive expression of endogenous galanin within degenerating cholinergic neurons, across the rostro-caudal axis of the PPN, by utilizing the lactacystin-induced rat model of PD. First, we show progressive neuronal death affecting nigral dopaminergic and PPN cholinergic neurons, reflecting that seen in PD patients, to facilitate use of this model for assessing the therapeutic potential of bioactive peptides. Next, stereological analyses of the lesioned brain hemisphere found that the number of PPN cholinergic neurons expressing galanin increased by 11%, compared to sham-lesioned controls, and increasing by a further 5% as the neurodegenerative process evolved. Galanin upregulation within cholinergic PPN neurons was most prevalent closest to the intra-nigral lesion site, suggesting that galanin upregulation in such neurons adapt intrinsically to neurodegeneration, to possibly neuroprotect. This is the first report on the extent and pattern of galanin expression in cholinergic neurons across distinct PPN subregions in both the intact rat CNS and lactacystin-lesioned rats. The findings pave the way for future work to target galanin signaling in the PPN, to determine the extent to which upregulated galanin expression could offer a viable treatment strategy for ameliorating PD symptoms associated with cholinergic degeneration.

Mitochondrial DNA changes in pedunculopontine cholinergic neurons in Parkinson disease.

In Parkinson disease (PD), mitochondrial dysfunction associates with nigral dopaminergic neuronal loss. Cholinergic neuronal loss co-occurs, particularly within a brainstem structure, the pedunculopontine nucleus (PPN). We isolated single cholinergic neurons from postmortem PPNs of aged controls and PD patients. Mitochondrial DNA (mtDNA) copy number and mtDNA deletions were increased significantly in PD patients compared to controls. Furthermore, compared to controls the PD patients had significantly more PPN cholinergic neurons containing mtDNA deletion levels exceeding 60%, a level associated with deleterious effects on oxidative phosphorylation. The current results differ from studies reporting mtDNA depletion in nigral dopaminergic neurons of PD patients. Ann Neurol 2017;82:1016-1021.

MutPred mutational load analysis shows mildly deleterious mitochondrial DNA variants are not more prevalent in Alzheimer's patients, but may be under-represented in healthy older individuals.

Mitochondrial DNA (mtDNA) association studies have been conducted for over a decade using the haplogroup (lineage) association method, but this frequently produces conflicting results. Here we analyzed complete mtDNA sequence data of Alzheimer's disease (AD) patients and aged controls, from the United Kingdom (UK) and the United States (US), using a new "mutational load" method. We calculated a pathogenicity score for each of the non-synonymous substitutions of the mtDNA sequences to produce a "total mutational load" for each sequence, and compared the mutational loads of cases and controls. Using these mutational load measures, we found no evidence to support the cumulative role of mtDNA variants as a susceptibility factor in AD; that is, AD patients (UK and US cohorts) did not have higher "mutational loads" than controls. However, the US aged controls, who are significantly older than the UK ones, with many showing evidence of being healthy and having good cognition in old age, had significantly lower "mutational loads". This finding suggests that low mtDNA mutational load is more prevalent in healthy older people.

The Cellular Diversity of the Pedunculopontine Nucleus: Relevance to Behavior in Health and Aspects of Parkinson's Disease.

The pedunculopontine nucleus (PPN) is a rostral brainstem structure that has extensive connections with basal ganglia nuclei and the thalamus. Through these the PPN contributes to neural circuits that effect cortical and hippocampal activity. The PPN also has descending connections to nuclei of the pontine and medullary reticular formations, deep cerebellar nuclei, and the spinal cord. Interest in the PPN has increased dramatically since it was first suggested to be a novel target for treating patients with Parkinson's disease who are refractory to medication. However, application of frequency-specific electrical stimulation of the PPN has produced inconsistent results. A central reason for this is that the PPN is not a heterogeneous structure. In this article, we review current knowledge of the neurochemical identity and topographical distribution of neurons within the PPN of both humans and experimental animals, focusing on studies that used neuronally selective targeting strategies to ascertain how the neurochemical heterogeneity of the PPN relates to its diverse functions in relation to movement and cognitive processes. If the therapeutic potential of the PPN is to be realized, it is critical to understand the complex structure-function relationships that exist here.

Pedunculopontine cell loss and protein aggregation direct microglia activation in parkinsonian rats.

We previously reported a loss of cholinergic neurons within the pedunculopontine tegmental nucleus (PPTg) in rats that had been intra-nigrally lesioned with the proteasomal inhibitor lactacystin, with levels of neuronal loss corresponding to that seen in the post-mortem pedunculopontine nucleus (PPN) of advanced Parkinson's disease (PD) patients. Here we reveal lower expression values of the acetylcholine synthesising enzyme, choline acetyltransferase, within the remaining PPTg cholinergic neurons of lesioned rats compared to sham controls. We further characterise this animal model entailing dopaminergic- and non-dopaminergic neurodegeneration by reporting on stereological counts of non-cholinergic neurons, to determine whether the toxin is neuro-type specific. Cell counts between lesioned and sham-lesioned rats were analysed in terms of the topological distribution pattern across the rostro-caudal extent of the PPTg. The study also reports somatic hypotrophy in the remaining non-cholinergic neurons, particularly on the side closest to the nigral lesion. The cytotoxicity affecting the PPTg in this rat model of PD involves overexpression and accumulation of alpha-synuclein (αSYN), affecting cholinergic and non-cholinergic neurons as well as microglia on the lesioned hemispheric side. We ascertained that microglia within the PPTg become fully activated due to the extensive neuronal damage and neuronal death resulting from a lactacystin nigral lesion, displaying a distinct rostro-caudal distribution profile which correlates with PPTg neuronal loss, with the added implication that lactacystin-induced αSYN aggregation might trigger neuronophagia for promoting PPTg cell loss. The data provide critical insights into the mechanisms underlying the lactacystin rat model of PD, for studying the PPTg in health and when modelling neurodegenerative disease.