Defective Presynaptic Choline Transport Underlies Hereditary Motor Neuropathy

The neuromuscular junction (NMJ) is a specialized synapse with a complex molecular architecture that provides for reliable transmission between the nerve terminal and muscle fiber. Using linkage analysis and whole-exome sequencing of DNA samples from subjects with distal hereditary motor neuropathy type VII, we identified a mutation in SLC5A7, which encodes the presynaptic choline transporter (CHT), a critical determinant of synaptic acetylcholine synthesis and release at the NMJ. This dominantly segregating SLC5A7 mutation truncates the encoded product just beyond the final transmembrane domain, eliminating cytosolic-C-terminus sequences known to regulate surface transporter trafficking. Choline-transport assays in both transfected cells and monocytes from affected individuals revealed significant reductions in hemicholinium-3-sensitive choline uptake, a finding consistent with a dominant-negative mode of action. The discovery of CHT dysfunction underlying motor neuropathy identifies a biological basis for this group of conditions and widens the spectrum of disorders that derive from impaired NMJ transmission. Our findings compel consideration of mutations in SLC5A7 or its functional partners in relation to unexplained motor neuronopathies

Haploinsufficiency of the E3 Ubiquitin Ligase C-Terminus of Heat Shock Cognate 70 Interacting Protein (CHIP) Produces Specific Behavioral Impairments

The multifunctional E3 ubiquitin ligase CHIP is an essential interacting partner of HSP70, which together promote the proteasomal degradation of client proteins. Acute CHIP overexpression provides neuroprotection against neurotoxic mitochondrial stress, glucocorticoids, and accumulation of toxic amyloid fragments, as well as genetic mutations in other E3 ligases, which have been shown to result in familial Parkinson's disease. These studies have created a great deal of interest in understanding CHIP activity, expression and modulation. While CHIP knockout mice have the potential to provide essential insights into the molecular control of cell fate and survival, the animals have been difficult to characterize in vivo due to severe phenotypic and behavioral dysfunction, which have thus far been poorly characterized. Therefore, in the present study we conducted a battery of neurobehavioral and physiological assays of adult CHIP heterozygotic (HET) mutant mice to provide a better understanding of the functional consequence of CHIP deficiency. We found that CHIP HET mice had normal body and brain weight, body temperature, muscle tone and breathing patterns, but do have a significant elevation in baseline heart rate. Meanwhile basic behavioral screens of sensory, motor, emotional and cognitive functions were normative. We observed no alterations in performance in the elevated plus maze, light-dark preference and tail suspension assays, or two simple cognitive tasks: novel object recognition and spontaneous alternation in a Y maze. Significant deficits were found, however, when CHIP HET mice performed wire hang, inverted screen, wire maneuver, and open field tasks. Taken together, our data indicate a clear subset of behaviors that are altered at baseline in CHIP deficient animals, which will further guide whole animal studies of the effects of CHIP dysregulation on cardiac function, brain circuitry and function, and responsiveness to environmental and cellular stress

Intracranial V. cholerae Sialidase Protects against Excitotoxic Neurodegeneration

Converging evidence shows that GD3 ganglioside is a critical effector in a number of apoptotic pathways, and GM1 ganglioside has neuroprotective and noötropic properties. Targeted deletion of GD3 synthase (GD3S) eliminates GD3 and increases GM1 levels. Primary neurons from GD3S−/− mice are resistant to neurotoxicity induced by amyloid-β or hyperhomocysteinemia, and when GD3S is eliminated in the APP/PSEN1 double-transgenic model of Alzheimer's disease the plaque-associated oxidative stress and inflammatory response are absent. To date, no small-molecule inhibitor of GD3S exists. In the present study we used sialidase from Vibrio cholerae (VCS) to produce a brain ganglioside profile that approximates that of GD3S deletion. VCS hydrolyzes GD1a and complex b-series gangliosides to GM1, and the apoptogenic GD3 is degraded. VCS was infused by osmotic minipump into the dorsal third ventricle in mice over a 4-week period. Sensorimotor behaviors, anxiety, and cognition were unaffected in VCS-treated mice. To determine whether VCS was neuroprotective in vivo, we injected kainic acid on the 25th day of infusion to induce status epilepticus. Kainic acid induced a robust lesion of the CA3 hippocampal subfield in aCSF-treated controls. In contrast, all hippocampal regions in VCS-treated mice were largely intact. VCS did not protect against seizures. These results demonstrate that strategic degradation of complex gangliosides and GD3 can be used to achieve neuroprotection without adversely affecting behavior

BMP9 Protects Septal Neurons from Axotomy-Evoked Loss of Cholinergic Phenotype

Cholinergic projection from the septum to the hippocampus is crucial for normal cognitive function and degeneration of cells and nerve fibers within the septohippocampal pathway contributes to the pathophysiology of Alzheimer's disease. Bone morphogenetic protein (BMP) 9 is a cholinergic differentiating factor during development both in vivo and in vitro.To determine whether BMP9 could protect the adult cholinergic septohippocampal pathway from axotomy-evoked loss of the cholinergic phenotype, we performed unilateral fimbria-fornix transection in mice and treated them with a continuous intracerebroventricular infusion of BMP9 for six days. The number of choline acetyltransferase (CHAT)-positive cells was reduced by 50% in the medial septal nucleus ipsilateral to the lesion as compared to the intact, contralateral side, and BMP9 infusion prevented this loss in a dose-dependent manner. Moreover, BMP9 prevented most of the decline of hippocampal acetylcholine levels ipsilateral to the lesion, and markedly increased CHAT, choline transporter CHT, NGF receptors p75 (NGFR-p75) and TrkA (NTRK1), and NGF protein content in both the lesioned and unlesioned hippocampi. In addition, BMP9 infusion reduced bilaterally hippocampal levels of basic FGF (FGF2) protein.These data indicate that BMP9 administration can prevent lesion-evoked impairment of the cholinergic septohippocampal neurons in adult mice and, by inducing NGF, establishes a trophic environment for these cells

Choline transporter gene variation is associated with attention-deficit hyperactivity disorder

The neurotransmitter acetylcholine (ACh) plays a critical role in brain circuits mediating motor control, attention, learning and memory. Cholinergic dysfunction is associated with multiple brain disorders including Alzheimer’s Disease, addiction, schizophrenia and Attention-Deficit Hyperactivity Disorder (ADHD). The presynaptic choline transporter (CHT, SLC5A7) is the major, rate-limiting determinant of ACh production in the brain and periphery and is consequently upregulated during tasks that require sustained attention. Given the contribution of central cholinergic circuits to the control of movement and attention, we hypothesized that functional CHT gene variants might impact risk for ADHD. We performed a case-control study, followed by family-based association tests on a separate cohort, of two purportedly functional CHT polymorphisms (coding variant Ile89Val (rs1013940) and a genomic SNP 3’ of the CHT gene (rs333229), affording both a replication sample and opportunities to reduce potential population stratification biases. Initial genotyping of pediatric ADHD subjects for two purportedly functional CHT alleles revealed a 2–3 fold elevation of the Val89 allele (n = 100; P = 0.02) relative to healthy controls, as well as a significant decrease of the 3’SNP minor allele in Caucasian male subjects (n = 60; P = 0.004). In family based association tests, we found significant overtransmission of the Val89 variant to children with a Combined subtype diagnosis (OR = 3.16; P = 0.01), with an increased Odds Ratio for a haplotype comprising both minor alleles. These studies show evidence of cholinergic deficits in ADHD, particularly for subjects with the Combined subtype, and, if replicated, may encourage further consideration of cholinergic agonist therapy in the disorder

From synapse to gene product: prolonged expression of c-fos induced by a single microinjection of carbachol in the pontomesencephalic tegmentum

It is not known how the brain modifies its regulatory systems in response to the application of a drug, especially over the long term of weeks and months. We have developed a model system approach to this question by manipulating cholinergic cell groups of the laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei in the pontomesencephalic tegmentum (PMT), which are known to be actively involved in the timing and quantity of rapid eye movement (REM) sleep. In a freely moving feline model, a single microinjection of the cholinergic agonist carbachol conjugated to a latex nanosphere delivery system into the caudolateral PMT elicits a long-term enhancement of one distinguishing phasic event of REM sleep, ponto-geniculo-occipital (PGO) waves, lasting 5 days but without any significant change in REM sleep or other behavioral state. Here, we test the hypothesis that cholinergic activation within the caudolateral PMT alters the postsynaptic excitability of the PGO network, stimulating the prolonged expression of c-fos that underlies this long-term PGO enhancement (LTPE) effect. Using quantitative Fos immunohistochemistry, we found that the number of Fos-immunoreactive (Fos-IR) neurons surrounding the caudolateral PMT injection site decreased sharply by postcarbachol day 03, while the number of Fos-IR neurons in the more rostral LDT/PPT increased >30-fold and remained at a high level following the course of LTPE. These results demonstrate a sustained c-fos expression in response to pharmacological stimulation of the brain and suggest that carbachol's acute effects induce LTPE via cholinergic receptors, with subsequent transsynaptic activation of the LDT/PPT maintaining the LTPE effect

Obstructive Sleep Apnea: Another Condition to Screen for in Women with Infertility

Women with obstructive sleep apnea (OSA) are at increased risk of poor pregnancy outcomes such as fetal growth restriction, hypertensive disorders of pregnancy, and gestational diabetes mellitus. Given this increased risk, we aimed to study the screening prevalence of OSA in women seeking fertility treatment. We performed a cross sectional study of patients presenting to a university-affiliated fertility clinic between March-April 2021. Patients were asked to complete OSA screening (STOP-BANG), anxiety screening (GAD-7), and depression screening (PHQ-2) questionnaires. 107 women completed the surveys. Mean age was 35.1 years and mean body mass index (BMI) was 25.7 kg/m2. Nine (8.4%) women screened positive for OSA using the STOP-BANG screening tool. Women who screened positive for OSA were more likely to be older (37.8 years vs. 34.7 years, p = 0.02) and have a higher BMI (42.6 kg/m2 vs. 27.4 kg/m2, p p = 0.006) and mild-severe anxiety (66.7% vs. 21.4%, p = 0.003) symptoms. 24.3% of the population had polycystic ovary syndrome (PCOS). Women with PCOS were more likely to screen positive for OSA (19.2% vs. 4.9%; p = 0.04). Despite this being a low-risk population of young women seeking fertility evaluation or treatment, 8% screened positive for OSA. Given the association between OSA and adverse pregnancy outcomes, our results underline the need to screen women seeking fertility treatment

Attenuated Vasodilator Effectiveness of Protease-Activated Receptor 2 Agonist in Heterozygous par2 Knockout Mice

Studies of homozygous PAR2 gene knockout mice have described a mix of phenotypic effects in vitro and in vivo. However, there have been few studies of PAR2 heterozygous (wild-type/knockout; PAR2-HET) mice. The phenotypes of many hemi and heterozygous transgenic mice have been described as intermediates between those of wild-type and knockout animals. In our study we aimed to determine the effects of intermediary par2 gene zygosity on vascular tissue responses to PAR2 activation. Specifically, we compared the vasodilator effectiveness of the PAR2 activating peptide 2-furoyl-LIGRLO-amide in aortas of wild-type PAR2 homozygous (PAR2-WT) and PAR2-HET mice. In myographs under isometric tension conditions, isolated aortic rings were contracted by alpha 1-adrenoeceptor agonist (phenylephrine), and thromboxane receptor agonist (U46619) and then relaxation responses by the additions of 2-furoyl-LIGRLO-amide, acetylcholine, and nitroprusside were recorded. A Schild regression analysis of the inhibition by a PAR2 antagonist (GB-83) of PAR2 agonist-induced aortic ring relaxations was used to compare receptor expression in PAR2-WT to PAR2-HET. PAR2 mRNA in aortas was measured by quantitative real-time PCR. In aortas contracted by either phenylephrine or U46619, the maximum relaxations induced by 2-furoyl-LIGRLO-amide were less in PAR2-HET than in the gender-matched PAR2-WT. GB-83 was 3- to 4-fold more potent for inhibition of 2fly in PAR2-HET than in PAR2-WT. PAR2 mRNA content of aortas from PAR2-HET was not significantly different than in PAR2-WT. Acetylcholine- and nitroprusside-induced relaxations of aortas from PAR2-HET were not significantly different than in PAR2-WT and PAR2 knockout. An interesting secondary finding was that relaxations induced by agonists of PAR2 and muscarinic receptors were larger in females than in males. We conclude that the lower PAR2-mediated responses in PAR2-HET aortas are consistent with evidence of a lower quantity of functional receptor expression, despite the apparently normal PAR2 mRNA content in PAR2-HET aortas