Premutation CGG repeat expansions (55-200 CGG repeats; preCGG) within the fragile X mental retardation 1 (FMR1) gene cause fragile X-associated tremor/ataxia syndrome (FXTAS). Defects in neuronal morphology and migration have been described in a preCGG mouse model. Mouse preCGG hippocampal neurons (170 CGG repeats) grown in vitro develop abnormal networks of clustered burst (CB) firing, as assessed by multielectrode array recordings and clustered patterns of spontaneous Ca(2+) oscillations, neither typical of wild-type (WT) neurons. PreCGG neurons have reduced expression of vesicular GABA and glutamate (Glu) transporters (VGAT and VGLUT1, respectively), and preCGG hippocampal astrocytes display a rightward shift on Glu uptake kinetics, compared with WT. These alterations in preCGG astrocytes and neurons are associated with 4- to 8-fold elevated Fmr1 mRNA and occur despite consistent expression of fragile X mental retardation protein levels at ∼50% of WT levels. Abnormal patterns of activity observed in preCGG neurons are pharmacologically mimicked in WT neurons by addition of Glu or the mGluR1/5 agonist, dihydroxyphenylglycine, to the medium, or by inhibition of astrocytic Glu uptake with dl-threo-β-benzyloxyaspartic acid, but not by the ionotropic Glu receptor agonists, α-2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid or N-methyl-d-aspartic acid. The mGluR1 (7-(hydroxyimino)cyclopropa [b]chromen-1a-carboxylate ethyl ester) or mGluR5 (2-methyl-6-(phenylethynyl)pyridine hydrochloride) antagonists reversed CB firing. Importantly, the acute addition of the neurosteroid allopregnanolone mitigated functional impairments observed in preCGG neurons in a reversible manner. These results demonstrate abnormal mGluR1/5 signaling in preCGG neurons, which is ameliorated by mGluR1/5 antagonists or augmentation of GABA(A) receptor signaling, and identify allopregnanolone as a candidate therapeutic lead.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a leading monogenic neurodegenerative disorder affecting premutation carriers of the fragile X (FMR1) gene. To investigate the underlying cellular neuropathology, we produced induced pluripotent stem cell-derived neurons from isogenic subclones of primary fibroblasts of a female premutation carrier, with each subclone bearing exclusively either the normal or the expanded (premutation) form of the FMR1 gene as the active allele. We show that neurons harboring the stably-active, expanded allele (EX-Xa) have reduced postsynaptic density protein 95 protein expression, reduced synaptic puncta density and reduced neurite length. Importantly, such neurons are also functionally abnormal, with calcium transients of higher amplitude and increased frequency than for neurons harboring the normal-active allele. Moreover, a sustained calcium elevation was found in the EX-Xa neurons after glutamate application. By excluding the individual genetic background variation, we have demonstrated neuronal phenotypes directly linked to the FMR1 premutation. Our approach represents a unique isogenic, X-chromosomal epigenetic model to aid the development of targeted therapeutics for FXTAS, and more broadly as a model for the study of common neurodevelopmental (e.g. autism) and neurodegenerative (e.g. Parkinsonism, dementias) disorders.

Premutation CGG repeat expansions (55–200 CGG repeats; preCGG) within the fragile X mental retardation 1 (FMR1) gene cause fragile X-associated tremor/ataxia syndrome (FXTAS) in humans. Defects in neuronal morphology, early migration, and electrophysiological activity have been described despite appreciable expression of FMRP in a preCGG knock-in (KI) mouse model. The triggers that initiate and promote preCGG neuronal dysfunction are not understood. The absence of FMRP in a Drosophila model of fragile X syndrome was shown to increase axonal transport of mitochondria. Here we show that dissociated hippocampal neuronal culture from preCGG KI mice (average 170 CGG repeats) express 42.6% of the FMRP levels and 3.8-fold higher Fmr1 mRNA than that measured in wild type neurons at 4 days in vitro. PreCGG hippocampal neurons show abnormalities in the number, mobility, and metabolic function of mitochondria at this early stage of differentiation. PreCGG hippocampal neurites contained significantly fewer mitochondria and greatly reduced mitochondria mobility. In addition, preCGG neurons had higher rates of basal oxygen consumption and proton leak. We conclude that deficits in mitochondrial trafficking and metabolic function occur despite the presence of appreciable FMRP expression and may contribute to the early pathophysiology in preCGG carriers and to the risk of developing clinical FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurological disorder that affects premutation carriers with 55-200 CGG-expansion repeats (preCGG) in FMR1, presenting with early alterations in neuronal network formation and function that precede neurodegeneration. Whether intranuclear inclusions containing DNA damage response (DDR) proteins are causally linked to abnormal synaptic function, neuronal growth and survival are unknown. In a mouse that harbors a premutation CGG expansion (preCGG), cortical and hippocampal FMRP expression is moderately reduced from birth through adulthood, with greater FMRP reductions in the soma than in the neurite, despite several-fold elevation of Fmr1 mRNA levels. Resting cytoplasmic calcium concentration ([Ca2+]i) in cultured preCGG hippocampal neurons is chronically elevated, 3-fold compared to Wt; elevated ROS and abnormal glutamatergic responses are detected at 14 DIV. Elevated µ-calpain activity and a higher p25/p35 ratio in the cortex of preCGG young adult mice indicate abnormal Cdk5 regulation. In support, the Cdk5 substrate, ATM, is upregulated by 1.5- to 2-fold at P0 and 6 months in preCGG brain, as is p-Ser1981-ATM. Bax:Bcl-2 is 30% higher in preCGG brain, indicating a greater vulnerability to apoptotic activation. Elevated [Ca2+]i, ROS, and DDR signals are normalized with dantrolene. Chronic [Ca2+]i dysregulation amplifies Cdk5-ATM signaling, possibly linking impaired glutamatergic signaling and DDR to neurodegeneration in preCGG brain.

Background: FMR1 CGG expansion repeats in the premutation range have not been linked to astrocyte pathophysiology. Results: Premutation cortical astrocytes display decreased Glu transporter expression/activity and enhanced asynchronous Ca 2ϩ oscillations. Conclusion: Glu transport and Ca 2ϩ signaling defects in premutation astrocytes could contribute to FXTAS neuropathology. Significance: Premutation astrocytes may have an etiological role in FXTAS neuropathology.

Barium currents through voltage-gated calcium (Ca2+) channels were studied in the small-cell lung carcinoma cell line NCI-H345 using patch clamp techniques. Pharmacological dissection of whole-cell barium currents revealed that 23% of the current was sensitive to nitrendipine, 35% to omega-conotoxin GVIA, and between 10 and 39% to omega-Aga-IVA. This implies that these cells express L-, N-, and P-type calcium channels. Only large cells expressed current that was sensitive to omega-Aga-IVA. The size dependency of this P-type channel expression may reflect the cell cycle stage. Cell-attached recordings revealed three unitary conductances: 5 to 6 pS, 10 to 12 pS, and 20 to 23 pS. The largest conductance channel (20-23 pS) was sensitive to Bay K 8644 and is presumed to represent L-type calcium channels. The frequency of observing the medium conductance channel (10-12 pS) was reduced by exposure to omega-conotoxin GVIA and may represent N-type channels. Incubation of cells with Lambert-Eaton myasthenic syndrome IgG for 24 to 48 hours removed up to 71% of the whole-cell current. Incubation with control human IgG (normal or myasthenia gravis) had no effect. Lambert-Eaton myasthenic syndrome IgG did not selectively target one "presynaptic" type of calcium channel, but rather appeared to target many of the calcium channel types that are expressed on small-cell lung carcinoma cells.

Tetramethylenedisulfotetramine (TETS) is a potent convulsant GABAA receptor blocker. Mice receiving a lethal dose of TETS (0.15 mg/kg i.p.) are rescued from death by a high dose of diazepam (5 mg/kg i.p.) administered shortly after the second clonic seizure (∼20 min post-TETS). However, this high dose of diazepam significantly impairs blood pressure and mobility, and does not prevent TETS-induced neuroinflammation in the brain. We previously demonstrated that TETS alters synchronous Ca2+ oscillations in primary mouse hippocampal neuronal cell cultures and that pretreatment with the combination of diazepam and allopregnanolone at concentrations having negligible effects individually prevents TETS effects on intracellular Ca2+ dynamics. Here, we show that treatment with diazepam and allopregnanolone (0.1 μM) 20 min after TETS challenge normalizes synchronous Ca2+ oscillations when added in combination but not when added singly. Similarly, doses (0.03–0.1 mg/kg i.p.) of diazepam and allopregnanolone that provide minimal protection when administered singly to TETS intoxicated mice increase survival from 10% to 90% when given in combination either 10 min prior to TETS or following the second clonic seizure. This therapeutic combination has negligible effects on blood pressure or mobility. Combined treatment with diazepam and allopregnanolone also decreases TETS-induced microglial activation. Diazepam and allopregnanolone have distinct actions as positive allosteric modulators of GABAA receptors that in combination enhance survival and mitigate neuropathology following TETS intoxication without the adverse side effects associated with high dose benzodiazepines. Combination therapy with a benzodiazepine and neurosteroid represents a novel neurotherapeutic strategy with potentially broad application.

Primary cultured hippocampal neurons (HN) form functional networks displaying synchronous Ca 21 oscillations (SCOs) whose patterns influence plasticity. Whether chemicals with distinct seizurogenic mechanisms differentially alter SCO patterns was investigated using mouse HN loaded with the Ca 21 indicator fluo-4-AM. Intracellular Ca 21 dynamics were recorded from 96 wells simultaneously in real-time using fluorescent imaging plate reader.