Methamphetamine decreases K+ channel function in human fetal astrocytes by activating the trace amine-associated receptor type-1.

Methamphetamine (Meth) is a potent and commonly abused psychostimulant. Meth alters neuron and astrocyte activity; yet the underlying mechanism(s) is not fully understood. Here we assessed the impact of acute Meth on human fetal astrocytes (HFAs) using whole-cell patch-clamping. We found that HFAs displayed a large voltage-gated K+ efflux (IKv ) through Kv /Kv -like channels during membrane depolarization, and a smaller K+ influx (Ikir ) via inward-rectifying Kir /Kir -like channels during membrane hyperpolarization. Meth at a 'recreational' (20 µM) or toxic/fatal (100 µM) concentration depolarized resting membrane potential (RMP) and suppressed IKv/Kv-like . These changes were associated with a decreased time constant (Ƭ), and mimicked by blocking the two-pore domain K+ (K2P )/K2P -like and Kv /Kv -like channels, respectively. Meth also diminished IKir/Kir-like , but only at toxic/fatal levels. Given that Meth is a potent agonist for the trace amine-associated receptor type-1 (TAAR1), and TAAR1-coupled cAMP/cAMP-activated protein kinase (PKA) cascade, we further evaluated whether the Meth impact on K+ efflux was mediated by this pathway. We found that antagonizing TAAR1 with N-(3-Ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl)benzamide (EPPTB) reversed Meth-induced suppression of IKv/Kv-like ; and inhibiting PKA activity by H89 abolished Meth effects on suppressing IKv/Kv-like . Antagonizing TAAR1 might also attenuate Meth-induced RMP depolarization. Voltage-gated Ca2+ currents were not detected in HFAs. These novel findings demonstrate that Meth suppresses IKv/Kv-like by facilitating the TAAR1/Gs /cAMP/PKA cascade and altering the kinetics of Kv /Kv -like channel gating, but reduces K2P /K2P -like channel activity through other pathway(s), in HFAs. Given that Meth-induced decrease in astrocytic K+ efflux through K2P /K2P -like and Kv /Kv -like channels reduces extracellular K+ levels, such reduction could consequently contribute to a decreased excitability of surrounding neurons. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Aging alters voltage-gated calcium channels in prefrontal cortex pyramidal neurons in the HIV brain.

We assessed firing and voltage-gated Ca2+ influx in medial prefrontal cortex (mPFC) pyramidal neurons from older (12 months old) HIV-1 transgenic (Tg) rats. We found that neurons from older Tg rats showed increased firing compared to non-Tg rats, but Ca2+ spikes were unchanged. However, stronger excitatory stimulation was needed to evoke Ca2+ spikes, which was associated with reduced mPFC Cav1.2 L-type Ca2+ channel (L-channel) protein. In contrast, L-channel protein was unaltered in younger (6-7 weeks old) Tg rats, which we previously found had enhanced neuronal Ca2+ influx. These studies demonstrate that aging alters HIV-induced Ca2+ channel dysfunction that affects mPFC activity.

Combined chronic blockade of hyper-active L-type calcium channels and NMDA receptors ameliorates HIV-1 associated hyper-excitability of mPFC pyramidal neurons.

Human Immunodeficiency Virus type 1 (HIV-1) infection induces neurological and neuropsychological deficits, which are associated with dysregulation of the medial prefrontal cortex (mPFC) and other vulnerable brain regions. We evaluated the impact of HIV infection in the mPFC and the therapeutic potential of targeting over-active voltage-gated L-type Ca(2+) channels (L-channel) and NMDA receptors (NMDAR), as modeled in HIV-1 transgenic (Tg) rats. Whole-cell patch-clamp recording was used to assess the membrane properties and voltage-sensitive Ca(2+) potentials (Ca(2+) influx) in mPFC pyramidal neurons. Neurons from HIV-1 Tg rats displayed reduced rheobase, spike amplitude and inwardly-rectifying K(+) influx, increased numbers of action potentials, and a trend of aberrant firing compared to those from non-Tg control rats. Neuronal hyper-excitation was associated with abnormally-enhanced Ca(2+) influx (independent of NMDAR), which was eliminated by acute L-channel blockade. Combined chronic blockade of over-active L-channels and NMDARs with open-channel blockers abolished HIV effects on spiking, aberrant firing and Ca(2+) potential half-amplitude duration, though not the reduced inward rectification. In contrast, individual chronic blockade of over-active L-channels or NMDARs did not alleviate HIV-induced mPFC hyper-excitability. These studies demonstrate that HIV alters mPFC neuronal activity by dysregulating membrane excitability and Ca(2+) influx through the L-channels. This renders these neurons more susceptible and vulnerable to excitatory stimuli, and could contribute to HIV-associated neuropathogenesis. Combined targeting of over-active L-channels/NMDARs alleviates HIV-induced dysfunction of mPFC pyramidal neurons, emphasizing a potential novel therapeutic strategy that may effectively decrease HIV-induced Ca(2+) dysregulation in the mPFC.

HIV-Induced Hyperactivity of Striatal Neurons Is Associated with Dysfunction of Voltage-Gated Calcium and Potassium Channels at Middle Age.

Despite combination antiretroviral therapy, HIV-associated neurocognitive disorders (HAND) occur in ~50% of people living with HIV (PLWH), which are associated with dysfunction of the corticostriatal pathway. The mechanism by which HIV alters the neuronal activity in the striatum is unknown. The goal of this study is to reveal the dysfunction of striatal neurons in the context of neuroHIV during aging. Using patch-clamping electrophysiology, we evaluated the functional activity of medium spiny neurons (MSNs), including firing, Ca2+ spikes mediated by voltage-gated Ca2+ channels (VGCCs), and K+ channel-mediated membrane excitability, in brain slices containing the dorsal striatum (a.k.a. the caudate-putamen) from 12-month-old (12mo) HIV-1 transgenic (HIV-1 Tg) rats. We also assessed the protein expression of voltage-gated Cav1.2/Cav1.3 L-type Ca2+ channels (L-channels), NMDA receptors (NMDAR, NR2B subunit), and GABAA receptors (GABAARs, ß2,3 subunit) in the striatum. We found that MSNs had significantly increased firing in 12mo HIV-1 Tg rats compared to age-matched non-Tg control rats. Unexpectedly, Ca2+ spikes were significantly reduced, while Kv channel activity was increased, in MSNs of HIV-1 Tg rats compared to non-Tg ones. The reduced Ca2+ spikes were associated with an abnormally increased expression of a shorter, less functional Cav1.2 L-channel form, while there was no significant change in the expression of NR2Bs or GABAARs. Collectively, the present study initially reveals neuroHIV-induced dysfunction of striatal MSNs in 12mo-old (middle) rats, which is uncoupled from VGCC upregulation and reduced Kv activity (that we previously identified in younger HIV-1 Tg rats). Notably, such striatal dysfunction is also associated with HIV-induced hyperactivity/neurotoxicity of glutamatergic pyramidal neurons in the medial prefrontal cortex (mPFC) that send excitatory input to the striatum (demonstrated in our previous studies). Whether such MSN dysfunction is mediated by alterations in the functional activity instead of the expression of NR2b/GABAAR (or other subtypes) requires further investigation.

Aging and HIV-1 alter the function of specific K+ channels in prefrontal cortex pyramidal neurons.

The medial prefrontal cortex (mPFC) is a key regulator of neurocognition. The glutamatergic pyramidal neurons are the predominant component of neurons in the mPFC. Aging and HIV profoundly alter the structure and function of mPFC pyramidal neurons, including, but are not limited to, dysregulation of NMDA receptors and voltage-gated calcium channels. Here we assessed the impact of aging and in vivo HIV exposure on the functional activity (firing) of mPFC pyramidal neurons mediated by voltage-gated K+ (Kv) channels and inwardly-rectifying K+ (Kir) channels using patch-clamp recording in rat brain slices ex vivo. We found that aging and HIV significantly affect firing in different manners by altering the activity of Kv and likely Kir channels, associated with changes in membrane properties and the mRNA levels of specific Kv channels. Evoked firing was significantly decreased in mPFC neurons of older (12 month, 12 m) rats compared to younger (6/7 week, 6/7wk) rats, regardless of HIV status. In contrast, firing was significantly increased in neurons from Tg rats compared to non-Tg rats, regardless of age. Aging/HIV-induced alterations in firing were mediated by dysfunctional Kv channels and Kir channels, which exhibit significant changes in their activity and/or expression induced by aging and HIV exposure in vivo. Collectively, these novel findings demonstrate that aging is associated with a significant decline of mPFC neuronal activity; while long-term HIV exposure in vivo could drive mPFC neurons from over-activation to loss of firing, which could ultimately exacerbate the decline of mPFC neuronal activity.

Long-term, homologous prolactin, administered through ectopic pituitary grafts, induces hypothalamic dopamine neuron differentiation in adult Snell dwarf mice.

Pituitary prolactin (PRL) secretion is inhibited by dopamine (DA) released into the portal circulation from hypothalamic tuberoinfundibular DA (TIDA) neurons. Ames (df/df) and Snell (dw/dw) dwarf mice lack PRL, GH, and TSH, abrogating feedback and resulting in a reduced hypophysiotropic TIDA population. In Ames df/df, ovine PRL administration for 30 d during early postnatal development increases the TIDA neuron number to normal, but 30 d PRL treatment of adult df/df does not. The present study investigated the effects of homologous PRL, administered via renal capsule pituitary graft surgery for 4 or 6 months, on hypothalamic DA neurons in adult Snell dw/dw mice using catecholamine histofluorescence, tyrosine hydroxylase immunocytochemistry, and bromodeoxyuridine immunocytochemistry. PRL treatment did not affect TIDA neuron number in normal mice, but 4- and 6-month PRL-treated dw/dw had significantly increased (P < or = 0.01) TIDA (area A12) neurons compared with untreated dw/dw. Snell dwarfs treated with PRL for 6 months had more (P < or = 0.01) TIDA neurons than 4-month PRL-treated dw/dw, but lower (P < or = 0.01) numbers than normal mice. Periventricular nucleus (area A14) neuron number was lower in dwarfs than in normal mice, regardless of treatment. Zona incerta (area A13) neuron number was unchanged among phenotypes and treatments. Prolactin was unable to induce differentiation of a normal-sized A14 neuron population in dw/dw. Bromodeoxyuridine incorporation was lower (P < or = 0.01) in 6-month PRL-treated normal mice than in 6-month PRL-treated dwarfs in the subventricular zone of the lateral ventricle and in the dentate gyrus, and lower (P < or = 0.05) in 4-month untreated dwarfs than in 4-month untreated normal mice in the median eminence and the periventricular area surrounding the third ventricle. Thus, a PRL-sensitive TIDA neuron population exists in adult Snell dwarf mice when replacement uses homologous hormone and/or a longer duration. This finding indicates that there is potential for neuronal differentiation beyond early developmental periods and suggests plasticity within the mature hypothalamus.

Targeting alpha-synuclein with a microRNA-embedded silencing vector in the rat substantia nigra: positive and negative effects.

BACKGROUND: Alpha-synuclein (SNCA) downregulation shows therapeutic potential for synucleinopathies, including Parkinson's disease (PD). Previously we showed that human (h)SNCA gene silencing using a short hairpin (sh)RNA in rat substantia nigra (SN) protects against a hSNCA-induced forelimb deficit, but not dopamine (DA) neuron loss. Furthermore, the shRNA increases cell death in vitro, but the same target sequence embedded in a microRNA30 transcript (mir30-hSNCA) does not. OBJECTIVE: Examine hSNCA gene silencing using mir30-hSNCA in vivo. METHODS: Rats were stereotaxically injected into one SN with adeno-associated virus serotype 2/8 (AAV)-hSNCA, AAV-hSNCA plus AAV-mir30-SNCA or AAV-hSNCA plus a control non-silencing mir30-embedded siRNA and DA neuron markers and associated behavior were examined. RESULTS: AAV2/8-mediated SN hSNCA expression induces a forelimb deficit and tyrosine hydroxylase-immunoreactive (TH-IR) neuron loss. hSNCA gene silencing using mir30-hSNCA protects against this forelimb deficit at 2 m and ameliorates TH-IR neuron loss. Striatal (ST) TH-IR fiber density and DA markers, assessed by western blot, are unaffected by AAV-hSNCA alone. Co-expression of either silencing vector reduces ST TH-IR fibers, panTH in SN and Ser40 phosphorylated TH in SN and ST, but does not affect vesicular monoamine transporter-2. However, hSNCA gene silencing promotes partial TH-IR fiber recovery by 2 m. Co-expression of either silencing vector also induces SN inflammation, although some recovery was observed by 2 m in hSNCA-silenced SN. CONCLUSION: hSNCA gene silencing with AAV-mir30-hSNCA has positive effects on forelimb behavior and SN DA neurons, which are compromised by inflammation and reduced TH expression, suggesting that AAV2/8-mir30-hSNCA-mediated gene silencing, although promising in vitro, is not a candidate for therapeutic translation for PD.

Comparable dimerization found in wildtype and familial Alzheimer's disease amyloid precursor protein mutants.

Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder marked by memory impairment and cognitive deficits. A major component of AD pathology is the accumulation of amyloid plaques in the brain, which are comprised of amyloid beta (Aß) peptides derived from the amyloidogenic processing of the amyloid precursor protein (AßPP) by ß- and γ-secretases. In a subset of patients, inheritance of mutations in the AßPP gene is responsible for altering Aß production, leading to early onset disease. Interestingly, many of these familial mutations lie within the transmembrane domain of the protein near the GxxxG and GxxxA dimerization motifs that are important for transmembrane interactions. As AßPP dimerization has been linked to changes in Aß production, it is of interest to know whether familial AßPP mutations affect full-length APP dimerization. Using bimolecular fluorescence complementation (BiFC), blue native gel electrophoresis, and live cell chemical cross-linking, we found that familial Alzheimer's disease (FAD) mutations do not affect full-length AßPP dimerization in transfected HEK293 and COS7 cells. It follows that changes in AßPP dimerization are not necessary for altered Aß production, and in FAD mutations, changes in Aß levels are more likely a result of alternative proteolytic processing.

Inclusion of a portion of the native SNCA 3'UTR reduces toxicity of human S129A SNCA on striatal-projecting dopamine neurons in rat substantia nigra.

Experimental models of Parkinson's disease (PD) created by aberrant expression of the alpha-synuclein (SNCA) coding region have been reported. However, noncoding regions function in normal physiology and recent in vitro studies have shown that microRNAs-7 and -153 regulate SNCA expression by binding the 3'UTR. Here, effects of different hSNCA forms were examined in vivo. Adult, male rats were injected into one substantia nigra (SN) with AAV-wtSNCA, AAV-S129A hSNCA, or AAV-S129D hSNCA either with or without a portion of the native 3'UTR. DA neurons in SN that maintained striatal (ST) projections at the end of treatment were retrogradely labeled by bilateral ST fluorogold (FG) injections and FG-positive DA neurons in SN were counted. At 5 weeks, hSNCA coding vectors reduced numbers of FG-positive neurons in injected SN compared with uninjected SN (wtSNCA, p = 0.05; S129A/D hSNCA, p = 0.01). At 7 and 9 weeks, wtSNCA- and S129D hSNCA-treated rats exhibited recovery, but S129A hSNCA-injected rats did not (p = 0.01). In contrast, numbers of FG-positive neurons were unaffected by hSNCA expression when the 3'UTR was included. When FG-positive neurons were expressed as the ratio of numbers in injected to uninjected sides, the S129A hSNCA coding vector resulted in the highest decrease at 9 weeks versus wtSNCA (p = 0.05) or S129D hSNCA (p = 0.01). Inclusion of the 3'UTR resulted in no significant differences in FG-positive neuron ratios. These data suggest that inclusion of the 3'UTR protects against S129A hSNCA-induced loss of nigrostriatal-projecting DA neurons in vivo and that mis-regulation of hSNCA expression and function at noncoding regions contribute to PD pathogenesis.

A microRNA embedded AAV α-synuclein gene silencing vector for dopaminergic neurons.

Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson's disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons.

An α-synuclein AAV gene silencing vector ameliorates a behavioral deficit in a rat model of Parkinson's disease, but displays toxicity in dopamine neurons.

Effects of silencing ectopically expressed hSNCA in rat substantia nigra (SN) were examined as a novel therapeutic approach to Parkinson's disease (PD). AAV-hSNCA with or without an AAV harboring a short-hairpin (sh)RNA targeting hSNCA or luciferase was injected into one SN. At 9weeks, hSNCA-expressing rats had reduced SN dopamine (DA) neurons and exhibited a forelimb deficit. AAV-shRNA-SNCA silenced hSNCA and protected against the forelimb deficit. However, AAV-shRNA-SNCA also led to DA neuron loss suggesting undesirable effects of chronic shRNA expression. Effects on nigrostriatal-projecting neurons were examined using a retrograde tract tracer. Loss of striatal-projecting DA neurons was evident in the vector injection site, whereas DA neurons outside this site were lost in hSNCA-expressing rats, but not in hSNCA-silenced rats. These observations suggest that high levels of shRNA-SNCA were toxic to DA neurons, while neighboring neurons exposed to lower levels were protected by hSNCA gene silencing. Also, data collected on DA levels suggest that neurons other than or in addition to nigrostriatal DA neurons contributed to protection of forelimb use. Our observations suggest that while hSNCA gene silencing in DA neurons holds promise as a novel PD therapy, further development of silencing technology is required.

Early postnatal administration of growth hormone increases tuberoinfundibular dopaminergic neuron numbers in Ames dwarf mice.

Hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons secrete dopamine, which inhibits pituitary prolactin (PRL) secretion. PRL has demonstrated neurotrophic effects on TIDA neuron development in PRL-, GH-, and TSH-deficient Ames (df/df) and Snell (dw/dw) dwarf mice. However, both PRL and PRL receptor knockout mice exhibit normal-sized TIDA neuron numbers, implying GH and/or TSH influence TIDA neuron development. The current study investigated the effect of porcine (p) GH on TIDA neuron development in Ames dwarf hypothalamus. Normal (DF/df) and dwarf mice were treated daily with pGH or saline beginning at 3 d of age for a period of 42 d. After treatment, brains were analyzed using catecholamine histofluorescence, tyrosine hydroxylase immunocytochemistry, and bromodeoxyuridine (BrdU) immunocytochemistry to detect BrdU incorporation. DF/df males and df/df treated with pGH experienced increased (P