Gabapentinoid Insensitivity after Repeated Administration is Associated with Down-Regulation of the α_2δ-1 Subunit in Rats with Central Post-Stroke Pain Hypersensitivity

The α_2δ-1 subunit of the voltage-gated Ca~(2+)channel(VGCC) is a molecular target of gabapentin(GBP), which has been used as a first-line drug for the relief of neuropathic pain. GBP exerts its anti-nociceptive effects by disrupting trafficking of the α_2δ-1 subunit to the presynaptic membrane, resulting in decreased neurotransmitter release. We previously showed that GBP has an antiallodynic effect in the first two weeks; but this is followed by insensitivity in the later stage after repeated administration in a rat model of central post-stroke pain(CPSP)hypersensitivity induced by intra-thalamic hemorrhage. To explore the mechanisms underlying GBP insensitivity, the cellular localization and time-course of expression of the α_2δ-1 subunit in both the thalamus and spinal dorsal horn were studied in the same model. We found that the α_2δ-1subunit was mostly localized in neurons, but not astrocytes and microglia. The level of α_2δ-1 protein increased in the first two weeks after injury but then decreased in the third week, when GBP insensitivity occurred. Furthermore, the α_2δ-1 down-regulation was likely caused by later neuronal loss in the injured thalamus through a mechanism other than apoptosis. In summary, the present results suggest that the GBP receptor α_2δ-1 is mainly expressed in thalamic neurons in which it is up-regulated in the early stage of CPSP but this is followed by dramatic down-regulation,which is likely associated with GBP insensitivity after long-term use.     

Involvement of the α<Subscript>1D</Subscript>-adrenergic Receptor in Methamphetamine-Induced Hyperthermia and Neurotoxicity in Rats

Methamphetamine (METH) is a psychostimulant that damages nigrostriatal dopaminergic terminals, primarily by enhancing dopamine and glutamate release. α₁-adrenergic receptor (AR) subtype involved in METH-induced neurotoxicity in rats was investigated using selective α₁-AR antagonists. METH neurotoxicity was evaluated by (1) measuring body temperature; (2) determining tyrosine hydroxylase (TH) immunoreactivity levels; (3) examining levels of dopamine and its metabolites; and (4) assessing glial fibrillary acidic protein (GFAP) and microglial immunoreactivity in the striatum. METH caused a decrease in dopamine and TH levels and induced hyperthermia which is an exacerbating factor of METH neurotoxicity. Concurrently, METH increased GFAP expression and the number of activated microglia. Pretreatment with prazosin, a nonselective α₁-AR antagonist, completely abolished METH-induced decrease in both dopamine and TH and caused a partial reduction in hyperthermia. Prazosin also prevented METH-induced increase in both GFAP expression and the number of activated microglia. In vivo microdialysis analysis revealed that prazosin, however, does not alter the METH-induced dopamine release in the striatum. The neuroprotective effects of prazosin could be mimicked by a selective α_1D antagonist, BMY 7378, but not by selective α_1A or α_1B antagonists. These results suggest that the α_1D-AR is involved in METH-induced hyperthermia and neurotoxicity in rats.     

Fast Inactivation of Ca<Subscript>V</Subscript>2.2 Channels Is Prevented by the Gβ<Subscript>1</Subscript> Subunit in Rat Sympathetic Neurons

Voltage-dependent regulation of Ca_V2.2 channels by G-proteins is performed by the β (Gβ) subunit. Most studies of regulation by G-proteins have focused on channel activation; however, little is known regarding channel inactivation. This study investigated inactivation of Ca_V2.2 channels in superior cervical ganglion neurons that overexpressed Gβ subunits. Ca_V2.2 currents were recorded by whole-cell patch clamping configuration. We found that the Gβ₁ subunit reduced inactivation, while Gβ₅ subunit did not alter at all inactivation kinetics compared to control recordings. Ca_V2.2 current decay in control neurons consisted of both fast and slow inactivation; however, Gβ₁-overexpressing neurons displayed only the slow inactivation. Fast inactivation was restored by a strong depolarization of Gβ₁-overexpressing neurons, therefore, through a voltage-dependent mechanism. The Gβ₁ subunit shifted the voltage dependence of inactivation to more positive voltages and reduced the fraction of Ca_V2.2 channels resting in the inactivated state. These results support that the Gβ₁ subunit inhibits the fast inactivation of Ca_V2.2 channels in SCG neurons. They explain the long-observed sustained Ca²⁺ current under G-protein modulation.     

The Neuropeptide Orexin-A Inhibits the GABA<Subscript>A</Subscript> Receptor by PKC and Ca<Superscript>2+</Superscript>/CaMKII-Dependent Phosphorylation of Its β<Subscript>1</Subscript> Subunit

Orexin-A and orexin-B (Ox-A, Ox-B) are neuropeptides produced by a small number of neurons that originate in the hypothalamus and project widely in the brain. Only discovered in 1998, the orexins are already known to regulate several behaviours. Most prominently, they help to stabilise the waking state, a role with demonstrated significance in the clinical management of narcolepsy and insomnia. Orexins bind to G-protein-coupled receptors (predominantly postsynaptic) of two subtypes, OX₁R and OX₂R. The primary effect of Ox-OXR binding is a direct depolarising influence mediated by cell membrane cation channels, but a wide variety of secondary effects, both pre- and postsynaptic, are also emerging. Given that inhibitory GABAergic neurons also influence orexin-regulated behaviours, crosstalk between the two systems is expected, but at the cellular level, little is known and possible mechanisms remain unidentified. Here, we have used an expression system approach to examine the feasibility, and nature, of possible postsynaptic crosstalk between Ox-A and the GABA_A receptor (GABA_AR), the brain’s main inhibitory neuroreceptor. When HEK293 cells transfected with OX₁R and the α₁, β₁, and γ_2S subunits of GABA_AR were exposed to Ox-A, GABA-induced currents were inhibited, in a calcium-dependent manner. This inhibition was associated with increased phosphorylation of the β₁ subunit of GABA_AR, and the inhibition could itself be attenuated by (1) kinase inhibitors (of protein kinase C and CaM kinase II) and (2) the mutation, to alanine, of serine 409 of the β₁ subunit, a site previously identified in phosphorylation-dependent regulation in other pathways. These results are the first to directly support the feasibility of postsynaptic crosstalk between Ox-A and GABA_AR, indicating a process in which Ox-A could promote phosphorylation of the β₁ subunit, reducing the GABA-induced, hyperpolarising current. In this model, Ox-A/GABA_AR crosstalk would cause the depolarising influence of Ox-A to be boosted, a type of positive feedback that could, for example, facilitate the ability to abruptly awake.     

Changes in the mRNA Levels of α<Subscript>2A</Subscript> and α<Subscript>2C</Subscript> Adrenergic Receptors in Rat Models of Parkinson’s Disease and <Emphasis Type="SmallCaps">l</Emphasis>-DOPA-Induced Dyskinesia

The changes in the mRNA levels of α_2A and α_2C adrenoceptors were investigated in unilateral 6-OHDA-lesioned rat model of Parkinson’s disease and l-DOPA-induced dyskinesia using in situ hybridization. In the untreated 6-OHDA-lesioned rats, α_2A expression was elevated in the locus coeruleus (160 ± 8% and 142 ± 8% in lesioned and unlesioned sides compared to the comparable side in sham-operated rats). Following long-term (21 days, twice daily) treatment with l-DOPA (25 mg/kg l-DOPA methyl ester plus benserazide 6.25 mg/kg) in 6-OHDA-lesioned rats, levels of α_2A adrenoceptor mRNA in the locus coeruleus were decreased, compared to the 6-OHDA-lesioned rats, returning to the levels of α_2A mRNA in the sham-operated rats. α_2A adrenoceptor expression was not changed in other brain regions in any treatment group. There was no change in α_2C expression in the rostral or caudal striatum in which the highest density of α_2C mRNA is present. In conclusion, the data presented in this study demonstrate an increase in α_2A adrenoceptor mRNA in the locus coeruleus in the 6-OHDA-lesioned rat model of Parkinson’s disease. In addition, the data show that repeated treatment with l-DOPA in 6-OHDA-lesioned rats, which induces dyskinesia, restores α_2A mRNA levels. These changes of α_2A mRNA expression, observed in the locus coeruleus, might be of importance to basal ganglia transmission and motor function.     

New molecular targets for antiepileptic drugs: α<Subscript>2</Subscript>δ, SV2A,and K<Subscript>v</Subscript>7/KCNQ/M potassium channels

Many currently prescribed antiepileptic drugs (AEDs) act via voltage-gated sodium channels, through effects on γ-aminobutyric acid-mediated inhibition, or via voltage-gated calcium channels. Some newer AEDs do not act via these traditional mechanisms. The molecular targets for several of these nontraditional AEDs have been defined using cellular electrophysiology and molecular approaches. Here, we describe three of these targets: α₂δ, auxiliary subunits of voltage-gated calcium channels through which the gabapentinoids gabapentin and pregabalin exert their anticonvulsant and analgesic actions; SV2A, a ubiquitous synaptic vesicle glycoprotein that may prepare vesicles for fusion and serves as the target for levetiracetam and its analog brivaracetam (which is currently in late-stage clinical development); and K_v7/KCNQ/M potassium channels that mediate the M-current, which acts a brake on repetitive firing and burst generation and serves as the target for the investigational AEDs retigabine and ICA-105665. Functionally, all of the new targets modulate neurotransmitter output at synapses, focusing attention on presynaptic terminals as critical sites of action for AEDs.     

Selective loss of P2Y<Subscript>2</Subscript> nucleotide receptor immunoreactivity is associated with Alzheimer’s disease neuropathology

The uridine nucleotide-activated P2Y(2), P2Y(4) and P2Y(6) receptors are widely expressed in the brain and are involved in many CNS processes, including those which malfunction in Alzheimer's disease (AD). However, the status of these receptors in the AD neocortex, as well as their putative roles in the pathogenesis of neuritic plaques and neurofibrillary tangles, remain unclear. In this study, we used immunoblotting to measure P2Y(2), P2Y(4) and P2Y(6) receptors in two regions of the postmortem neocortex of neuropathologically assessed AD patients and aged controls. P2Y(2) immunoreactivity was found to be selectively reduced in the AD parietal cortex, while P2Y(4) and P2Y(6) levels were unchanged. In contrast, all three receptors were preserved in the occipital cortex, which is known to be minimally affected by AD neuropathology. Furthermore, reductions in parietal P2Y(2) immunoreactivity correlated both with neuropathologic scores and markers of synapse loss. These results provide a basis for considering P2Y(2) receptor changes as a neurochemical substrate of AD, and point towards uridine nucleotide-activated P2Y receptors as novel targets for disease-modifying AD pharmacotherapeutic strategies.     

Expression of α<Subscript>2</Subscript>-macroglobulin,neutrophil elastase,and interleukin-1α differs in early-stage and late-stage atherosclerotic lesions in the arteries of the circle of Willis

Different types of atherosclerotic (AS) lesions can be distinguished histologically and represent different stages of AS plaque development. Late-stage lesions more frequently develop complications such as plaque rupture and thrombosis with vessel occlusion than early AS lesions. To clarify whether protective, destructive, and inflammatory proteins are differentially expressed in early-stage and late-stage AS plaques we examined the proteinase inhibitor α₂-macroglobulin (A2M), the neutrophil elastase (NE)—an enzyme degrading elastin and collagen fibers—and the proinflammatory protein interleukin-1α (IL-1α) in all types of AS plaques in the arteries of the circle of Willis from 78 human autopsy cases of both genders (61–91 years of age). Paraffin sections of AS plaques were immunostained with antibodies directed against A2M, NE and IL-1α. In initial AS lesions A2M was found, whereas NE and IL-1α were absent. NE and IL-1α became detectable as soon as a significant number of macrophages occurred within AS lesions. With increasing histopathological type of AS lesions, a marked increase of the area of the plaque exhibiting NE and IL-1α was observed. The area which exhibits A2M in AS plaques, on the other hand, did not vary significantly between the different stages. Thus, our results indicate a disproportionately high increase of the destructive enzyme NE and the proinflammatory protein IL-1α in relation to A2M with the progression of the grade of AS lesions pointing to the transgression of the protective capacity of A2M by NE and IL-1α in late-stage plaques. Therefore, our findings support the hypothesis that NE-induced tissue damage in late-stage AS plaques contributes to the development of plaque rupture and subsequent thrombosis.     

Ovariectomy combined with amyloid β<Subscript>1-42</Subscript> impairs memory by decreasing acetylcholine release and α7nAChR Expression without induction of apoptosis in the hippocampus CA1 neurons of rats

In this study, the effect of ovariectomy and amyloid P_1-42 (Aβ_1-42)on eight-armed radial maze performance, acetylcholine (ACh) release, α7nACh receptor (α7nAChR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and apoptosis of CA1 neurons in the dorsal hippocampus were investigated in rat. The results showed that the dorsal hippocampus of sham rats contains 136.7 ± 16.7 to 160.4 ± 21.1 fmol/μl ACh, and respective 201 ± 22.9 and 416.6 ± 66.3 expression of mRNA for α7nAChR and GAPDH. Ovariectomy alone, after 4 weeks, did not impair memory, and neither induced apoptosis nor changed the basal ACh release. On the other hand, Aβ_1-42 (600 pmol/10 μl/body/day i.c.v. for 7 days) impaired memory, an effect characterized by increased error choices and reduced (50–59%) ACh release, but only with slight apoptosis. Moreover, ovariectomy combined with Aβ_1-42 induced memory impairment characterized by decreased numbers of correct choices and increased numbers of errors. This effect was accompanied by a decrease of the basal ACh level (67%), α7nAChR mRNA expression (52%) and α7nAChR/GAPDH ratio (44%) without induction of apoptosis in the dorsal hippocampus. The high K⁺-evoked ACh release was not altered in ovariectomized rats, but was decreased by Aβ_1-42 (43%) and ovariectomy + Aβ_1-42 (80%). These results suggest that ovariectomy-induced hormonal deprivation after 4 weeks, when accompanied by Aβ_1-42 accumulation in the dorsal hippocampus, could impair memory by decreasing ACh release and α7nAChR expression without inducing apoptosis in the CA1 field of the dorsal hippocampus.     

Bilateral Olfactory Mucosa Damage Induces the Disappearance of Olfactory Glomerulus and Reduces the Expression of Extrasynaptic α5GABA<Subscript>A</Subscript>Rs in the Hippocampus in Early Postnatal Sprague Dawley Rats

Chloroform-induced olfactory mucosal degeneration has been reported in adult rats following gavage. We used fixed-point chloroform infusions on different postnatal days (PNDs) to investigate the effects of early olfactory bilateral deprivation on the main olfactory bulbs in Sprague Dawley rats. The experimental groups included rats infused with chloroform (5 μl) or saline (sham, 5 μl) on PNDs 3 and 8, and rats not receiving infusions (control) (n?=?6 in all groups). Rats receiving chloroform on PND 3 showed significant hypoevolutism when compared to those in other groups (P?<?0.05). There was a complete disappearance and a significant reduction in the size of olfactory glomeruli in the PND 3 and 8 groups, respectively, when compared to the respective sham groups. Rats receiving chloroform on PND 3 had significant memory impairment (P?<?0.01) and increased levels of learned helplessness (P?<?0.05), as measured using the Morris water maze and tail suspension tests, respectively. GABA_A receptor alpha5 subunit (α5GABA_AR) expression in hippocampal neurons was significantly lower in rats receiving chloroform on PND 3 than in rats in other groups (P?<?0.01), as measured using immunohistochemistry and polymerase chain reaction. There was thus a critical period for the preservation of regenerative ability in olfactory receptor neurons, during which damage and olfactory deprivation led to altered rhinencephalon structure and disappearance of olfactory glomeruli, which induced hypoevolutism. Olfactory deprivation after the critical period had no significant effect on olfactory receptor neuron regeneration, leading to reduced developmental and behavioral effects in Sprague Dawley rats.     

Increased infections with β-blocker use in ischemic stroke,a β<Subscript>2</Subscript>-receptor mediated process?

Strokes promote immunosuppression, partially from increased sympathetic activity. Altering sympathetic drive with β-blockers has variably been shown to improve stroke outcomes. This study adds to this literature using propensity score matching to limit confounding and by examining the effects of selective and non-selective β-blockers. Prospective data from acute ischemic stroke admissions at a single center from July 2010–June 2015 were analyzed. Outcomes included infection (urinary tract infection [UTI], pneumonia, or bacteremia), discharge modified Rankin Score (mRS), and in-hospital death. Any selective and non-selective β-blocker use during the first 3 days of admission were investigated with propensity score matching. A sensitivity analysis was also performed. This study included 1431 admissions. Any β-blocker use was associated with increased infections (16.4 vs. 10.7%, p = 0.030). Non-selective β-blocker use was associated with increased infections (18.9 vs. 9.7%, p = 0.005) and UTIs (13.0 vs. 5.5%, p = 0.009). Selective β-blocker use was not associated with infection. There were no associations between β-blocker use and in-hospital death or discharge mRS. In the sensitivity analysis, the association between non-selective β-blocker use and urinary tract infections persisted (12.5 vs. 4.2%, p = 0.044). No associations with death or mRS were found. Early β-blocker use after ischemic stroke may increase the risk of infection but did not change disability or mortality risk. The mechanism may be mediated by β₂-adrenergic receptor antagonism given the different effects seen with selective versus non-selective β-blocker use.     

Allosteric Modulator Desformylflustrabromine Relieves the Inhibition of α2β2 and α4β2 Nicotinic Acetylcholine Receptors by β-Amyloid<Subscript>1–42</Subscript> Peptide

Nicotinic acetylcholine receptors (nAChRs) are pentameric transmembrane proteins that belong to the cys-loop ligand-gated ion channel family. These receptors are widely expressed in the brain and implicated in the pathophysiology of many neurological conditions, including Alzheimer’s disease (AD), where typical symptoms include the loss of cognitive function and dementia. The presence of extracellular neuritic plaques composed of β amyloid (Aβ_1–42) peptide is a characteristic feature of AD. Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) for α4β2 nAChRs since it increases peak ACh responses without inducing a response on its own. Previously, the effect of dFBr on the α2β2 nAChR subtype was not known. The action of dFBr was tested on α2β2 receptors expressed in Xenopus oocytes. It was found that dFBr is also a PAM for the α2β2 receptor. Next we tested whether dFBr had any effect on the previously known block of both the α4β2 and α2β2 receptors by Aβ_1–42. We found that the functional blockade of ACh-induced currents in oocytes expressing α4β2 and α2β2 receptors by Aβ_1–42 was prevented by dFBr. We conclude that dFBr is a positive allosteric modulator for both α4β2 and α2β2 subtypes of nAChRs and that it also relieves the blockade of these receptors by Aβ_1–42. This study demonstrates that PAMs for the non-α7 nAChRs have the potential to develop into clinically applicable drugs for AD and other disorders.     

Involvement of postsynaptic α1- and α2-adrenoceptors in the flexor reflex activity in the spinal Rats

Summary Clonidine (0.2 mg/kg i.v.) and St 587 (1 mg/kg i.v.), selective agonsists of ₂- and ₁-adrenoceptors, respectively, increased the flexor reflex amplitude in the spinal rat. Yohimbine and rauwolscine, ₂-adrenoceptor antagonists, inhibited dose-dependently the effect of clonidine but not of St 587. However, prazosin and clozapine, ₁-adrenoceptor antagonists, diminished the action of both agonists in a dose-dependent manner. After central chemosympathectomy caused by 6-OH-DA or DSP-4 the response to clonidine did not differ from that in the sham-operated animals. In 6-OH-DA treated rats yohimbine (1 mg/kg i.v.) antagonized the effect of clonidine to the same extent as it did in unlesioned animals. It is concluded that the results are functional evidence that ₁- and ₂-adrenoceptors are present in the rat spinal cord, and stimulation of each receptor increases flexor reflex activity.     

Increased Cerebrospinal Fluid F<Subscript>2</Subscript>-Isoprostanes are Associated with Aging and Latent Alzheimer’s Disease as Identified by Biomarkers

Alzheimer’s disease (AD) is a common age-related chronic illness with latent, prodrome, and fully symptomatic dementia stages. Increased free radical injury to regions of brain is one feature of prodrome and dementia stages of AD; however, it also is associated with advancing age. This raises the possibility that age-related free radical injury to brain might be caused in part or in full by latent AD. We quantified free radical injury in the central nervous system with cerebrospinal fluid (CSF) F₂-isoprostanes (IsoPs) in 421 clinically normal individuals and observed a significant increase over the adult human lifespan (P < 0.001). Using CSF amyloid (A) β₄₂ and tau, we defined normality using results from 28 clinically normal individuals <50 years old, and then stratified 74 clinically normal subjects ≥60 years into those with CSF that had normal CSF Aβ₄₂ and tau (n = 37); abnormal CSF Aβ₄₂ and tau, the biomarker signature of AD (n = 24); decreased Aβ₄₂ only (n = 4); or increased tau only (n = 9). Increased CSF F₂-IsoPs were present in clinically normal subjects with the biomarker signature of AD (P < 0.05) and those subjects with increased CSF tau (P < 0.001). Finally, we analyzed the relationship between age and CSF F₂-IsoPs for those clinically normal adults with normal CSF (n = 37) and those with abnormal CSF Aβ₄₂ and/or tau (n = 37); only those with normal CSF demonstrated a significant increase with age (P < 0.01). These results show that CSF F₂-IsoPs increased across the human lifespan and that this age-related increase in free radical injury to brain persisted after culling those with laboratory evidence of latent AD.     

Interference of α-Synuclein Uptake by Monomeric β-Amyloid<Subscript>1–40</Subscript> and Potential Core Acting Site of the Interference

Increasing evidence suggests an important role of alpha-synuclein (α-Syn) in the pathogenesis of Parkinson’s disease (PD). The inter-neuronal spread of α-Syn via exocytosis and endocytosis has been proposed as an explanation for the neuropathological findings of PD in sub-clinical and clinical phases. Therefore, interfering the uptake of α-Syn by neurons may be an important step in slowing or modifying the propagation of the disease. The purposes of our study were to investigate if the uptake of α-Syn fibrils can be specifically interfered with monomeric β-Amyloid_1–40 (Aβ₄₀) and to characterise the core acting site of interference. Using a radioisotope-labelled uptake assay, we found an 80 % uptake reduction of α-Syn fibrils in neurons interfered with monomeric Aβ₄₀, but not β-Amyloid_1–42 (Aβ₄₂) as compared to controls. This finding was further confirmed by enzyme-linked immunosorbent assay (ELISA) with α-Syn uptake reduced from about 80 % (Aβ₄₂) to about 20 % (Aβ₄₀) relative to controls. To define the region of Aβ₄₀ peptide capable of the interference, we explored shorter peptides with less amino acid residues from both the C-terminus and N-terminus. We found that the interference effect was preserved if amino acid residue was trimmed to position 11 (from N-terminus) and 36 (from C-terminus), but dropped off significantly if residues were trimmed beyond these positions. We therefore deduced that the “core acting site” lies between amino acid residue positions 12–36. These findings suggest α-Syn uptake can be interfered with monomeric Aβ₄₀ and that the core acting site of interference might lie between amino acid residue positions 12–36.     

Role of dopamine D<Subscript>3</Subscript> and serotonin 5-HT<Subscript>1A</Subscript> receptors in <Emphasis Type="SmallCaps">l</Emphasis>-DOPA-induced dyskinesias and effects of sarizotan in the 6-hydroxydopamine-lesioned rat model of Parkinson’s disease

Sarizotan, a 5-HT_1A agonist with additional affinity for D₃ and D₄ receptors, has been demonstrated to have anti-dyskinetic effects. The mechanism by which these effects occur is not clear. Using unilateral 6-hydroxydopamine-lesioned rats that received chronic intraperitoneal (ip) administration of l-3,4-dihydroxyphenylalanine (l-DOPA) we investigated the involvement of D₃ and 5-HT_1A receptors in the effects of sarizotan on contraversive circling and abnormal involuntary movements (AIMs). Before sensitization by chronic l-DOPA treatment (12.5 with 3.25 mg/kg benserazide ip, twice daily for 21 days), no effect of the selective D₃ agonist, PD128907 (1 or 3 mg/kg ip), or the selective D₃ antagonist, GR103691 (0.5 or 1.5 mg/kg ip), was observed. Treatment with sarizotan (1 or 5 mg/kg ip) dose-dependently inhibited the l-DOPA-induced contraversive turning and AIMs. In co-treatment with the 5-HT_1A antagonist, WAY100635 (1 mg/kg ip), sarizotan failed to affect this behaviour, confirming the prominent 5-HT_1A receptor-mediated mechanism of action. In the presence of PD128907 (3 mg/kg ip), the effects of sarizotan on contraversive turning, locomotive dyskinesia and axial dystonia, but not on orolingual and forelimb dyskinesia, were blocked. On its own, PD128907 had no effect on the behavioural effects of l-DOPA except that it tended to reduce orolingual and forelimb dyskinesia. GR103691 had no effect on its own or in combination with sarizotan. These data identify an involvement of D₃ receptors in the action of sarizotan on some, but not all l-DOPA-induced motor side effects. This selective involvement is in contrast to the more general involvement of 5-HT_1A receptors in the anti-dyskinetic effects of sarizotan.     

Insulin-Like Growth Factor-1 Alleviates Expression of Aβ<Subscript>1–40</Subscript> and α-, β-, and γ-Secretases in the Cortex and Hippocampus of APP/PS1 Double Transgenic Mice

To examine the effect of subcutaneous injection of insulin-like growth factor-1 (IGF-1) on the expression of the amyloid protein (Aβ_1–40), α-secretase (ADAM10), β-secretase (BACE1), and γ-secretase (PS1) in APP/PS1 double transgenic mice. APP/PS1 double transgenic mice and wild-type mice were divided into wild-type group, wild-type therapy group, transgenome group, and transgenic therapy group. Subcutaneous injection of IGF-1 (50 μg/kg day) was administered once daily to the wild-type therapy group and transgenic therapy group for 8 weeks, respectively. The expression of the Aβ_1–40 in the cortex and hippocampus was detected by immunohistochemistry 8 weeks after administration. The levels of Aβ_1–40, DAM10, BACE1, and PS1 were analysed by Western blot. The expression of the Aβ_1–40 in the cortex of the gene therapy group was significantly lower than that of the transgenome group (p?<?0.05). In APP/PS1 double transgenic mice, BACE1 expression was markedly higher in both the hippocampus (p?<?0.001, p?=?0.00009) and the cortex (p?=?0.001), compared to that of the wild-type mice. The treatment of IGF-1 markedly reduced ADAM10 expression in the hippocampus in both transgenic mice and wild-type mice (p?<?0.05), whereas the treatment mainly decreased BACE1 expression in transgenic mice but not in the wild-type mice (p?<?0.05). No significant differences in PS1 levels were detected in all groups. IGF decreased Aβ_1–40 over-expression in the cortex and hippocampus and might inhibit the damage induced by Aβ_1–40 in APP/PS1 double transgenic mice. Our study suggests that IGF-1 should inhibit Aβ production through α-secretase and β-secretase but not γ-secretase.     

Tau protein and beta-amyloid<Subscript>1-42</Subscript> CSF levels in different phenotypes of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder with highly heterogeneous clinical manifestations. This fact has prompted many attempts to divide PD patients into clinical subgroups. This could lead to a better recognition of pathogenesis, improving targeted treatment and the prognosis of PD patients. The aim of the present study was to obtain cerebrospinal fluid (CSF) samples in PD patients and to search for a relationship between neurodegenerative CSF markers (tau protein, beta-amyloid_1-42 and index tau protein/beta-amyloid_1-42) and the clinical subtypes. PD patients were divided into three subgroups: early disease onset (EDO), tremor-dominant PD (TD-PD), and non-tremor dominant PD (NT-PD) according to the previously published classification. Neurodegenerative markers in the CSF were assessed in these three groups of patients suffering from PD (EDO-17, TD-15, NT-16 patients) and in a control group (CG) of 19 patients suffering from non-degenerative neurological diseases and 18 patients with Alzheimer’s disease (AD). The NT-PD patients were found to have significantly higher levels of CSF tau protein and index tau/beta than the control subjects and other Parkinsonian subgroups, but no significant differences in these markers were found between AD and NT-PD patients. In the context of more rapid clinical progression and more pronounced neuropathological changes in the NT-PD patient group, our results corroborate the opinion that CSF level of tau protein may be regarded as a potential laboratory marker of the presence and severity of neurodegeneration.     

Co-expression of β Subunits with the Voltage-Gated Sodium Channel Na<Subscript>V</Subscript>1.7: the Importance of Subunit Association and Phosphorylation and Their Effects on Channel Pharmacology and Biophysics

The voltage-gated sodium ion channel Na_V1.7 is crucial in pain signaling. We examined how auxiliary β2 and β3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human Na_V1.7α subunit was co-expressed with either β2 or β3 subunits in HEK-293 cells. The β2 subunits and the Na_V1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the β2 subunits did not change the biophysical properties of the channel. In contrast, β3 subunit was clearly associated with Na_V1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated β3 subunit was associated with the Na_V1.7α. Electrophysiological characterisation revealed that the β3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the β3 subunit reduced the susceptibility of Na_V1.7α to several sodium channel blockers. In addition, we assessed the functional effect of Na_V1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of β subunits with Na_V1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with β2. The β3 subunit significantly influences the function of Na_V1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of Na_V1.7 channel in pain signaling.     

The role of phospholipase A<Subscript>2</Subscript> in neuronal homeostasis and memory formation: implications for the pathogenesis of Alzheimer’s disease

Summary. Phospholipase A₂ (PLA₂) is a key enzyme in cerebral phospholipid metabolism. Preliminary post-mortem studies have shown that PLA₂ activity is decreased in frontal and parietal areas of the AD brain, which is in accordance with recent ³¹P-Magnetic Resonance Spectroscopy evidence of reduced phospholipid turnover in the pre-frontal cortex of moderately demented AD patients. Such abnormality may also be observed in peripheral cells, and reduced PLA₂ activity in platelet membranes of AD patients, and correlates with the severity of dementia. In rat hippocampal slices, PLA₂ has been implicated in mechanisms of synaptic plasticity. In adult rats, the stereotaxic injection of PLA₂ inhibitors in the CA1 area of hippocampus impaired, in a dose-dependent manner, the formation of short- and long-term memory. Additionally, such inhibition resulted in a reduction of the fluidity of hippocampal membranes. In primary cultures of cortical and hippocampal neurons, the inhibition of PLA₂ precluded neurite outgrowth, and the sustained inhibition of the enzyme in mature cultures lead to loss of viability. Taken together, these findings reinforce the involvement of PLA₂ enzymes in neurodevelopment and neurodegeneration processes, and further suggest that reduced PLA₂ activity, probably reducing membrane phospholipids breakdown, may contribute to the memory impairment in AD.