Impaired Mitochondrial Respiration in REM-Sleep Behavior Disorder: A Biomarker of Parkinson's Disease?

BACKGROUND: Idiopathic rapid eye movement (REM) sleep behavior disorder (iRBD) is associated with prodromal Parkinson's disease (PD), but the mechanisms linking phenoconversion of iRBD to PD have not yet been clarified. Considering the association between mitochondrial dysfunction and sleep disturbances in PD, we explored mitochondrial activity in fibroblasts derived from iRBD patients to identify a biochemical profile that could mark the presence of impending neurodegeneration. METHODS: The study involved 28 participants, divided into three groups: patients diagnosed with iRBD, PD patients converted from iRBD (RBD-PD), and healthy controls. We performed a comprehensive assessment of mitochondrial function, including an examination of mitochondrial morphology, analysis of mitochondrial protein expression levels by western blot, and measurement of mitochondrial respiration using the Seahorse XFe24 analyzer. RESULTS: In basal conditions, mitochondrial respiration did not differ between iRBD and control fibroblasts, but when cells were challenged with a higher energy demand, iRBD fibroblasts exhibited a significant (P = 0.006) drop in maximal and spare respiration compared to controls. Interestingly, RBD-PD patients showed the same alterations with a further significant reduction in oxygen consumption linked to adenosine triphosphate production (P = 0.032). Moreover, RBD-PD patients exhibited a significant decrease in protein levels of complexes III (P = 0.02) and V (P = 0.002) compared to controls, along with fragmentation of the mitochondrial network. iRBD patients showed similar, but milder alterations. CONCLUSIONS: Altogether, these findings suggest that mitochondrial dysfunctions in individuals with iRBD might predispose to worsening of the bioenergetic profile observed in RBD-PD patients, highlighting these alterations as potential predictors of phenoconversion to PD. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Ambroxol as a disease-modifying treatment to reduce the risk of cognitive impairment in GBA-associated Parkinson's disease: a multicentre, randomised, double-blind, placebo-controlled, phase II trial. The AMBITIOUS study protocol.

Background: Heterozygous mutations in the GBA gene, encoding the lysosomal enzyme ß-glucocerebrosidase (GCase), are the most frequent genetic risk factor for Parkinson's disease (PD). GBA-related PD (GBA-PD) patients have higher risk of dementia and reduced survival than non-carriers. Preclinical studies and one open-label trial in humans demonstrated that the chaperone ambroxol (ABX) increases GCase levels and modulates α-synuclein levels in the blood and cerebrospinal fluid (CSF). Methods and analysis: In this multicentre, double-blind, placebo-controlled, phase II clinical trial, we randomise patients with GBA-PD in a 1:1 ratio to either oral ABX 1.2 g/day or placebo. The duration of treatment is 52 weeks. Each participant is assessed at baseline and weeks 12, 26, 38, 52 and 78. Changes in the Montreal Cognitive Assessment score and the frequency of mild cognitive impairment and dementia between baseline and weeks 52 are the primary outcome measures. Secondary outcome measures include changes in validated scales/questionnaires assessing motor and non-motor symptoms. Neuroimaging features and CSF neurodegeneration markers are used as surrogate markers of disease progression. GCase activity, ABX and α-synuclein levels are also analysed in blood and CSF. A repeated-measures analysis of variance will be used for elaborating results. The primary analysis will be by intention to treat. Ethics and dissemination: The study and protocols have been approved by the ethics committee of centres. The study is conducted according to good clinical practice and the Declaration of Helsinki. The trial findings will be published in peer-reviewed journals and presented at conferences. Trial registration numbers: NCT05287503, EudraCT 2021-004565-13.

Neuroprotective and Symptomatic Effects of Cannabidiol in an Animal Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta, leading to classical PD motor symptoms. Current therapies are purely symptomatic and do not modify disease progression. Cannabidiol (CBD), one of the main phytocannabinoids identified in Cannabis Sativa, which exhibits a large spectrum of therapeutic properties, including anti-inflammatory and antioxidant effects, suggesting its potential as disease-modifying agent for PD. The aim of this study was to evaluate the effects of chronic treatment with CBD (10 mg/kg, i.p.) on PD-associated neurodegenerative and neuroinflammatory processes, and motor deficits in the 6-hydroxydopamine model. Moreover, we investigated the potential mechanisms by which CBD exerted its effects in this model. CBD-treated animals showed a reduction of nigrostriatal degeneration accompanied by a damping of the neuroinflammatory response and an improvement of motor performance. In particular, CBD exhibits a preferential action on astrocytes and activates the astrocytic transient receptor potential vanilloid 1 (TRPV1), thus, enhancing the endogenous neuroprotective response of ciliary neurotrophic factor (CNTF). These results overall support the potential therapeutic utility of CBD in PD, as both neuroprotective and symptomatic agent.

GBA Mutations Influence the Release and Pathological Effects of Small Extracellular Vesicles from Fibroblasts of Patients with Parkinson's Disease.

Heterozygous mutations in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), are the strongest known genetic risk factor for Parkinson's disease (PD). The molecular mechanisms underlying the increased PD risk and the variable phenotypes observed in carriers of different GBA mutations are not yet fully elucidated. Extracellular vesicles (EVs) have gained increasing importance in neurodegenerative diseases since they can vehiculate pathological molecules potentially promoting disease propagation. Accumulating evidence showed that perturbations of the endosomal-lysosomal pathway can affect EV release and composition. Here, we investigate the impact of GCase deficiency on EV release and their effect in recipient cells. EVs were purified by ultracentrifugation from the supernatant of fibroblast cell lines derived from PD patients with or without GBA mutations and quantified by nanoparticle tracking analysis. SH-SY5Y cells over-expressing alpha-synuclein (α-syn) were used to assess the ability of patient-derived small EVs to affect α-syn expression. We observed that defective GCase activity promotes the release of EVs, independently of mutation severity. Moreover, small EVs released from PD fibroblasts carrying severe mutations increased the intra-cellular levels of phosphorylated α-syn. In summary, our work shows that the dysregulation of small EV trafficking and alpha-synuclein mishandling may play a role in GBA-associated PD.

Profiling the Biochemical Signature of GBA-Related Parkinson's Disease in Peripheral Blood Mononuclear Cells.

BACKGROUND: GBA mutations are the commonest genetic risk factor for Parkinson's disease (PD) and also impact disease progression. OBJECTIVE: The objective of this study was to define a biochemical profile that could distinguish GBA-PD from non-mutated PD. METHODS: 29 GBA-PD, 37 non-mutated PD, and 40 controls were recruited; α-synuclein levels in plasma, exosomes, and peripheral blood mononuclear cells were analyzed, GCase and main GCase-related lysosomal proteins in peripheral blood mononuclear cells were measured. RESULTS: Assessment of plasma and exosomal α-synuclein levels did not allow differentiation between GBA-PD and non-mutated PD; conversely, measurements in peripheral blood mononuclear cells clearly distinguished GBA-PD from non-mutated PD, with the former group showing significantly higher α-synuclein levels, lower GCase activity, higher LIMP-2, and lower Saposin C levels. CONCLUSION: We propose peripheral blood mononuclear cells as an easily accessible and manageable model to provide a distinctive biochemical profile of GBA-PD, potentially useful for patient stratification or selection in clinical trials. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Effects of Circadian Phase Tailored Light Therapy on Sleep, Mood, and Cognition in Alzheimer's Disease: Preliminary Findings in a Pivotal Study.

It is shown that the circadian system is affected in patients with Alzheimer's disease (AD) even at an early stage of the disease and that such dysfunction may be detrimental to sleep, mood, and cognitive functioning. Light is a strong central modulator of the circadian rhythms and is potentially beneficial to mood and cognitive functioning via a direct effect or indirectly via its modulating effects on circadian rhythms. This study focuses on tracking the effect of light therapy on sleep quality, mood, and cognition in AD of mild/moderate severity. We performed a single-blind randomized controlled trial to investigate the effects of a light therapy treatment tailored to the individual circadian phase as measured by dim light melatonin onset (DLMO). Such a treatment induced an objective circadian phase shift consistent with the melatonin phase response curve to light exposure, led to a shortening of the phase angle DLMO-falling asleep time, and was associated with an improvement in subjective sleep quality and cognitive performance.

Neuroprotective effects of lignan 7-hydroxymatairesinol (HMR/lignan) in a rodent model of Parkinson's disease.

OBJECTIVES: Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNc). The proinflammatory response can occur early in the disease, contributing to nigrostriatal degeneration. Identification of the new molecules, which are able to slow down the degenerative process associated with PD, represents one of the main interests. Recently, natural polyphenols, especially lignans, have raised attention for their anti-inflammatory, antioxidant, and estrogenic activity at a peripheral level. The aim of this study was to evaluate the central effects of chronic treatment with lignan 7-hydroxymatairesinol (HMR/lignan) on neurodegenerative, neuroinflammatory processes and motor deficits induced by a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA) in rats to evaluate the potential neuroprotective properties of this compound. METHODS: Sprague-Dawley male rats underwent lignan (10 mg/kg) or vehicle treatment (oral) for 4 wk starting from the day of 6-OHDA injection. The degree of nigrostriatal damage was evaluated by immunohistochemistry. Moreover, we performed a quantitative and qualitative assessment of neuroinflammatory process, including phenotypic polarization of microglia and astrocytes. The motor performance was assessed by behavioral tests. RESULTS: We demonstrated that chronic treatment with HMR/lignan was able to slow down the progression of degeneration of striatal dopaminergic terminals in a rat model of PD, with a consequent improvement in motor performance. Nevertheless, the anti-inflammatory effect of HMR/lignan observed in SNc was not sufficient to protect dopaminergic cells bodies. CONCLUSION: These results suggest intriguing properties of HMR/lignan at neuroprotective and symptomatic levels in the context of PD.

Evening melatonin timing secretion in real life conditions in patients with Alzheimer disease of mild to moderate severity.

BACKGROUND: Circadian dysfunction is thought to take part in the pathogenesis of sleep disorders in Alzheimer's disease (AD) and in AD pathophysiology itself. OBJECTIVE: Our study aims to calculate dim light melatonin onset (DLMO) secretion in order to define the circadian phase in patients with AD at an early stage of the disease. METHODS: Twenty-one patients (M/F: 11/10; mean age 74.1 ± 5.4 years; mean disease duration 3.4 ± 1.6 years) with a diagnosis of AD and 17 healthy controls (HC; M/F: 10/7; mean age 67.47 ± 3.8 years) were investigated for subjective nocturnal sleep quality and chronotype, for DLMO and quantitative aspects of the evening melatonin secretion by means of a 5-point in-home evening melatonin saliva test. RESULTS: Subjective sleep quality score on the Pittsburgh Sleep Quality Index questionnaire (PSQI) above 5 (p = 0.24), insomnia frequency as measured by Sleep Condition Indicator Questionnaire (p = 0.823) and the subjective chronotype according to Morning Evening Questionnaire (MEQ) scores distribution (p = 0.464) did not differ between AD and HC. However, DLMO occurred significantly later (55 min; p = 0.028), and melatonin secretion following DLMO was significantly decreased in AD patients compared to HC. CONCLUSION: Initial evening secretion of melatonin proves to be delayed and mildly impaired in patients with a mild/moderate form of Alzheimer disease while patients' subjective sleep parameters and chronotype are reported to be similar to those of HC. These results indicate that subclinical altered patterns of melatonin secretion occur in subjects with AD at an early stage of the disease.

The Exosomal/Total α-Synuclein Ratio in Plasma Is Associated With Glucocerebrosidase Activity and Correlates With Measures of Disease Severity in PD Patients.

Intensive research efforts in the field of Parkinson's disease (PD) are focusing on identifying reliable biomarkers which possibly help physicians in predicting disease onset, diagnosis, and progression as well as evaluating the response to disease-modifying treatments. Given that abnormal alpha-synuclein (α-syn) accumulation is a primary component of PD pathology, this protein has attracted considerable interest as a potential biomarker for PD. Alpha-synuclein can be detected in several body fluids, including plasma, where it can be found as free form or in association with exosomes, small membranous vesicles secreted by virtually all cell types. Together with α-syn accumulation, lysosomal dysfunctions seem to play a central role in the pathogenesis of PD, given the crucial role of lysosomes in the α-syn degradation. In particular, heterozygous mutations in the GBA1 gene encoding lysosomal enzyme glucocerebrosidase (GCase) are currently considered as the most important risk factor for PD. Different studies have found that GCase deficiency leads to accumulation of α-syn; whereas at the same time, increased α-syn may inhibit GCase function, thus inducing a bidirectional pathogenic loop. In this study, we investigated whether changes in plasma total and exosome-associated α-syn could correlate with disease status and clinical parameters in PD and their relationship with GCase activity. We studied 39 PD patients (mean age: 65.2 ± 8.9; men: 25), without GBA1 mutations, and 33 age-matched controls (mean age: 61.9 ± 6.2; men: 15). Our results showed that exosomes from PD patients contain a greater amount of α-syn compared to healthy subjects (25.2 vs. 12.3 pg/mL, p < 0.001) whereas no differences were found in plasma total α-syn levels (15.7 vs. 14.8 ng/mL, p = 0.53). Moreover, we highlighted a significant increase of plasma exosomal α-syn/total α-syn ratio in PD patients (1.69 vs. 0.89, p < 0.001), which negatively correlates with disease severity (p = 0.014). Intriguingly, a significant inverse correlation between GCase activity and this ratio in PD subjects was found (p = 0.006). Additional and large-scale studies comparing GCase activity and pathological protein levels will be clearly needed to corroborate these data and determine whether the association between key players in the lysosomal system and α-syn can be used as diagnostic or prognostic biomarkers for PD.

Influence of Estrogen Modulation on Glia Activation in a Murine Model of Parkinson's Disease.

Epidemiological data suggest a sexual dimorphism in Parkinson disease (PD), with women showing lower risk of developing PD. Vulnerability of the nigrostriatal pathway may be influenced by exposure to estrogenic stimulation throughout fertile life. To further address this issue, we analyzed the progression of nigrostriatal damage, microglia and astrocyte activation and microglia polarization triggered by intrastriatal injection of dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in male, female and ovariectomized (OVX) mice, as well as in OVX mice supplemented with 17ßestradiol (OVX+E). Animals were sacrificed at different time points following 6-OHDA injection and brain sections containing striatum and substantia nigra pars compacta (SNc) underwent immunohistochemistry for tyrosine hydroxylase (TH) (dopaminergic marker), immunofluorescence for IBA1 and GFAP (markers of microglia and astrocyte activation, respectively) and triple immunoflorescent to identify polarization of microglia toward the cytotoxic M1 (DAPI/IBA1/TNFα) or cytoprotective M2 (DAPI/IBA1/CD206) phenotype. SNc damage induced by 6-OHDA was significantly higher in OVX mice, as compared to all other experimental groups, at 7 and 14 days after surgery. Astrocyte activation was higher in OVX mice with respect the other experimental groups, at all time points. Microglial activation in the SNc was detected at earlier time points in male, female and OVX+E, while in OVX mice was detected at all time-points. Microglia polarization toward the M2, but not the M1, phenotype was detected in female and OVX+E mice, while the M1 phenotype was observed only in male and OVX mice. Our results support the protective effects of estrogens against nigrostriatal degeneration, suggesting that such effects may be mediated by an interaction with microglia, which tend to polarize preferentially toward an M2, cytoprotective phenotype in the presence of intense estrogenic stimulation.

Complex Changes in the Innate and Adaptive Immunity Accompany Progressive Degeneration of the Nigrostriatal Pathway Induced by Intrastriatal Injection of 6-Hydroxydopamine in the Rat.

We investigated changes in innate and adaptive immunity paralleling the progressive nigrostriatal damage occurring in a neurotoxic model of Parkinson's disease (PD) based on unilateral infusion of 6-hydroxydopamine (6-OHDA) into the rat striatum. A time-course analysis was conducted to assess changes in morphology (activation) and cell density of microglia and astrocytes, microglia polarization (M1 vs. M2 phenotype), lymphocyte infiltration in the lesioned substantia nigra pars compacta (SNc), and modifications of CD8+ and subsets of CD4+ T cell in peripheral blood accompanying nigrostriatal degeneration. Confirming previous results, we observed slightly different profiles of activation for astrocytes and microglia paralleling nigral neuronal loss. For astrocytes, morphological changes and cell density increases were mostly evident at the latest time points (14 and 28 days post-surgery), while moderate microglia activation was present since the earliest time point. For the first time, in this model, we described the time-dependent profile of microglia polarization. Activated microglia clearly expressed the M2 phenotype in the earlier phase of the experiment, before cell death became manifest, gradually shifting to the M1 phenotype as SNc cell death started. In parallel, a reduction in the percentage of circulating CD4+ T regulatory (Treg) cells, starting as early as day 3 post-6-OHDA injection, was detected in 6-OHDA-injected rats. Our data show that nigrostriatal degeneration is associated with complex changes in central and peripheral immunity. Microglia activation and polarization, Treg cells, and the factors involved in their cross-talk should be further investigated as targets for the development of therapeutic strategies for disease modification in PD.

Intracarotid Infusion of Mesenchymal Stem Cells in an Animal Model of Parkinson's Disease, Focusing on Cell Distribution and Neuroprotective and Behavioral Effects.

UNLABELLED: Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for Parkinson's disease (PD) and systemic administration of these cells has been tested in preclinical and clinical studies. However, no information on survival and actual capacity of MSCs to reach the brain has been provided. In this study, we evaluated homing of intraarterially infused rat MSCs (rMSCs) in the brain of rats bearing a 6-hydroxydopamine (6-OHDA)-induced lesion of the nigrostriatal tract, to establish whether the toxin-induced damage is sufficient to grant MSC passage across the blood-brain barrier (BBB) or if a transient BBB disruption is necessary. The rMSC distribution in peripheral organs and the effects of cell infusion on neurodegenerative process and motor deficits were also investigated. rMSCs were infused 14 days after 6-OHDA injection. A hyperosmolar solution of mannitol was used to transiently permeabilize the BBB. Behavioral impairment was assessed by adjusting step test and response to apomorphine. Animals were sacrificed 7 and 28 days after cell infusion. Our work shows that appreciable delivery of rMSCs to the brain of 6-OHDA-lesioned animals can be obtained only after mannitol pretreatment. A notable percentage of infused cells accumulated in peripheral organs. Infusion of rMSCs did not modify the progression of 6-OHDA-induced damage or the motor impairment at the stepping test, but induced progressive normalization of the pathological response (contralateral turning) to apomorphine administration. These findings suggest that many aspects should be further investigated before considering any translation of MSC systemic administration into the clinical setting for PD treatment. SIGNIFICANCE: This study demonstrates that mesenchymal stem cells infused through the carotid artery do not efficiently cross the blood-brain barrier in rats with a Parkinson's disease-like degeneration of nigrostriatal neurons, unless a permeabilizing agent (e.g., mannitol) is used. The infusion did not reduce the neuronal damage and associated motor impairment, but abolished the motor abnormalities these animals typically show when challenged with a dopaminergic agonist. Therefore, although arterially infused mesenchymal stem cells did not show neurorestorative effects in this study's Parkinson's disease model, they appeared to normalize the pathological responsiveness of striatal neurons to dopaminergic stimulation. This capability should be further explored in future studies.

Ambroxol-induced rescue of defective glucocerebrosidase is associated with increased LIMP-2 and saposin C levels in GBA1 mutant Parkinson's disease cells.

Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD). Defective GCase has been reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect. To further explore this issue, we investigated GCase and elements supporting GCase function and trafficking in fibroblasts from sporadic PD patients--with or without heterozygous GBA1 mutations--and healthy subjects, in basal conditions and following in vitro exposure to ambroxol. We assessed protein levels of GCase, lysosomal integral membrane protein-2 (LIMP-2), which mediates GCase trafficking to lysosomes, GCase endogenous activator saposin (Sap) C and parkin, which is involved in degradation of defective GCase. We also measured activities of GCase and cathepsin D, which cleaves Sap C from precursor prosaposin. GCase activity was reduced in fibroblasts from GBA1-mutant patients and ambroxol corrected this defect. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups, while LIMP-2 levels were increased only in GBA1-mutant PD fibroblasts. Parkin levels were slightly increased only in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Our study confirms that GCase activity is deficient in fibroblasts of GBA1-mutant PD patients and that ambroxol corrects this defect. The drug increased Sap C and LIMP-2 protein levels, without interfering with parkin. These results confirm that chemical chaperone ambroxol modulates lysosomal markers, further highlighting targets that may be exploited for innovative PD therapeutic strategies.

Bioenergetic and proteolytic defects in fibroblasts from patients with sporadic Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a complex disease and the current interest and focus of scientific research is both investigating the variety of causes that underlie PD pathogenesis, and identifying reliable biomarkers to diagnose and monitor the progression of pathology. Investigation on pathogenic mechanisms in peripheral cells, such as fibroblasts derived from patients with sporadic PD and age/gender matched controls, might generate deeper understanding of the deficits affecting dopaminergic neurons and, possibly, new tools applicable to clinical practice. METHODS: Primary fibroblast cultures were established from skin biopsies. Increased susceptibility to the PD-related toxin rotenone was determined with apoptosis- and necrosis-specific cell death assays. Protein quality control was evaluated assessing the efficiency of the Ubiquitin Proteasome System (UPS) and protein levels of autophagic markers. Changes in cellular bioenergetics were monitored by measuring oxygen consumption and glycolysis-dependent medium acidification. The oxido-reductive status was determined by detecting mitochondrial superoxide production and oxidation levels in proteins and lipids. RESULTS: PD fibroblasts showed higher vulnerability to necrotic cell death induced by complex I inhibitor rotenone, reduced UPS function and decreased maximal and rotenone-sensitive mitochondrial respiration. No changes in autophagy and redox markers were detected. CONCLUSIONS: Our study shows that increased susceptibility to rotenone and the presence of proteolytic and bioenergetic deficits that typically sustain the neurodegenerative process of PD can be detected in fibroblasts from idiopathic PD patients. Fibroblasts might therefore represent a powerful and minimally invasive tool to investigate PD pathogenic mechanisms, which might translate into considerable advances in clinical management of the disease.

Single or combined treatment with L-DOPA and quinpirole differentially modulate expression and phosphorylation of key regulatory kinases in neuroblastoma cells.

In the past decades, the clinical use of dopamine agonists has expanded from adjunct therapy in patients with a deteriorating response to L-3,4-dihydroxyphenylalanine (L-DOPA) to monotherapy for the treatment of early PD. Dopamine agonists provide their antiparkinsonian benefit through stimulation of brain postsynaptic type 2 dopamine receptors that exert their effect through classical cAMP-dependent mechanisms, as well as cAMP-independent cellular signaling cascades, including the Akt/glycogen synthase kinase 3 (GSK3) pathway. Alterations of Akt/GSK3 have been observed and may contribute to the neurodegenerative processes and the development of L-DOPA-induced dyskinesia. The effects L-DOPA and quinpirole, a dopamine agonist, on the two key regulatory kinases, Akt and GSK3, were evaluated in neuroblastoma cell line. L-DOPA and dopamine agonist dose-dependently and differentially modulated Akt and GSK3 expression and phosphorylation when added alone or combined. The combined treatment inverted or potentiated the modulatory properties of the single compound. The drug- and concentration-dependent balance of dopamine receptor stimulation over auto-oxidation may distinctively modulate GSK3 isoforms and Akt. Our results indicate that particular attention must be given to drug concentration and combination when multiple therapies are applied for the clinical treatment of PD patients.

Adhesion molecules as potential targets for neuroprotection in a rodent model of Parkinson's disease.

Cell adhesion molecules might play an important role in the inflammatory mechanisms associated with neurodegeneration. We have previously observed, in rats, that subcutaneous injection of complete Freund's adjuvant (CFA), a pro-inflammatory agent that induces a peripheral inflammatory stimulus, reduces the nigrostriatal degeneration and microglial activation caused by stereotaxic injection of 6-hydroxydopamine (6-OHDA). Here we further investigated the effects of CFA in 6-OHDA-lesioned rats by evaluating the expression of selected adhesion molecules, both at central and peripheral levels. Male, Sprague-Dawley rats received a subcutaneous injection of CFA followed, 10 days later, by intrastriatal injection of 6-OHDA. Animals were sacrificed at various time points and changes affecting intercellular (ICAM-1), vascular (VCAM-1), platelet endothelial (PECAM-1) and neural (NCAM-1) cell adhesion molecules were analyzed in striatum, ventral midbrain (containing the substantia nigra) and sera. Our results confirmed the protective effect of systemic CFA on 6-OHDA-induced nigrostriatal degeneration. Injection of 6-OHDA increased striatal ICAM-1 and PECAM-1 expression, while opposite changes (decreased expression) were detected in the ventral midbrain, particularly for VCAM-1 and NCAM-1. Pretreatment with CFA counteracted these changes. Nigrostriatal degeneration also affected peripheral immune function, with lesioned animals showing increased sPECAM levels with respect to intact animals. Also in this case, CFA pretreatment blocked the 6-OHDA induced increase of sPECAM. Our findings confirm that a pre-existing, peripheral pro-inflammatory condition reduces the neuroinflammatory response and associated neurodegeneration provoked by centrally-administered 6-OHDA, with a mechanism that seems to involve selected adhesion molecules. The link between peripheral and central immune responses may, therefore, represent a target for new therapeutic strategies aimed at reducing the neuroinflammatory component associated with neurodegeneration.

The breakpoint identified in a balanced de novo translocation t(7;9)(p14.1;q31.3) disrupts the A-kinase (PRKA) anchor protein 2 gene (AKAP2) on chromosome 9 in a patient with Kallmann syndrome and bone anomalies.

We report the molecular characterization of a patient with Kallmann syndrome and bone anomalies bearing a balanced de novo translocation t(7;9)(p14.1;q31.3) which completely disrupts the A-kinase anchor protein 2 gene (AKAP2) on chromosome 9. In order to investigate the role of AKAP2 in the pathogenesis of the disease, we analyzed the expression of Akap2 in mouse embryos. The expression pattern was consistent with the phenotype observed and mAkap2 was actually found in the olfactory bulb and in the cartilagineous structures of the embryo. Since AKAP2 is supposed to bind and compartmentalize the PKA, we also analyzed the distribution and quantity of PKA in limphoblastoid cell lines of the patient compared with a control; these experiments did not demonstrate any differences between the cell lines. Furthermore a collection of 98 DNA samples from sporadic Kallmann patients was screened for mutations in this gene. The analysis revealed two different sequence variations observed in two patients but not in 200 control chromosomes: since they have been detected also in the unaffected mother of one of the two patients we can assume that they are rare polymorphisms, although we cannot exclude that they represent mutations with incomplete penetrance. Our findings suggest that the complex phenotype with Kallmann syndrome and bone anomalies observed in our patient could be the result of the interruption of the AKAP2 gene. However, a position effect mediated by the translocation could not be excluded. The screening of AKAP2 in other Kallmann patients will be necessary to elucidate its role in the pathogenesis of the disease.

The Kallmann syndrome gene homolog in C. elegans is involved in epidermal morphogenesis and neurite branching.

Kallmann syndrome is an inherited disorder defined by the association of anosmia and hypogonadism, owing to impaired targeting and migration of olfactory axons and gonadotropin-releasing hormone secreting neurons. The gene responsible for the X-linked form of Kallmann syndrome, KAL-1, encodes a secreted protein of still elusive function. It has been proposed that KAL-1 might be involved in some aspects of olfactory axon guidance. However, the unavailability of a mouse model, and the difficulties in studying cellular and axonal migration in vertebrates have hampered an understanding of its function. We have identified the C. elegans homolog, kal-1, and document its function in vivo. We show that kal-1 is part of a mechanism by which neurons influence migration and adhesion of epidermal cells undergoing morphogenesis during ventral enclosure and male tail formation. We also show that kal-1 affects neurite outgrowth in vivo by modulating branching. Finally, we find that human KAL-1 cDNA can compensate for the loss of worm kal-1 and that overexpression of worm or human KAL-1 cDNAs in the nematode results in the same phenotypes. These data indicate functional conservation between the human and nematode proteins and establish C. elegans as a powerful animal in which to investigate KAL function in vivo. Our findings add a new player to the set of molecules, which appear to underlie both morphogenesis and axonal/neuronal navigation in vertebrates and invertebrates.