Q134R: Small chemical compound with NFAT inhibitory properties improves behavioral performance and synapse function in mouse models of amyloid pathology

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging-related Alzheimer's disease (AD)-like pathology. However, concerns over adverse effects have slowed the transition of common CN-inhibiting drugs to the clinic for the treatment of AD and AD-related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN-mediated dephosphorylation of a non-NFAT target, either in vivo, or in vitro. Acute (= 3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild-type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal A beta plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders

Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in A\u3cem\u3eβ\u3c/em\u3e-Bearing Mice

Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer\u27s disease (AD). Astrocytes are a primary defense against hyperexcitability, but their functional phenotype during AD is poorly understood. Here, we found that activated astrocytes in the 5xFAD mouse model were strongly associated with proteolysis of the protein phosphatase calcineurin (CN) and the elevated expression of the CN-dependent transcription factor nuclear factor of activated T cells 4 (NFAT4). Intrahippocampal injections of adeno-associated virus vectors containing the astrocyte-specific promoter Gfa2 and the NFAT inhibitory peptide VIVIT reduced signs of glutamate-mediated hyperexcitability in 5xFAD mice, measured in vivo with microelectrode arrays and ex vivo brain slices, using whole-cell voltage clamp. VIVIT treatment in 5xFAD mice led to increased expression of the astrocytic glutamate transporter GLT-1 and to attenuated changes in dendrite morphology, synaptic strength, and NMDAR-dependent responses. The results reveal astrocytic CN/NFAT4 as a key pathologic mechanism for driving glutamate dysregulation and neuronal hyperactivity during AD

Q134R: Small Chemical Compound with NFAT Inhibitory Properties Improves Behavioral Performance and Synapse Function in Mouse Models of Amyloid Pathology

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging-related Alzheimer\u27s disease (AD)-like pathology. However, concerns over adverse effects have slowed the transition of common CN-inhibiting drugs to the clinic for the treatment of AD and AD-related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN-mediated dephosphorylation of a non-NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild-type mice (3–4 months old) infused with oligomeric Aβ peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild-type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aβ plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders

Inhibition of Soluble Tumor Necrosis Factor Ameliorates Synaptic Alterations and Ca2+ Dysregulation in Aged Rats

The role of tumor necrosis factor α (TNF) in neural function has been investigated extensively in several neurodegenerative conditions, but rarely in brain aging, where cognitive and physiologic changes are milder and more variable. Here, we show that protein levels for TNF receptor 1 (TNFR1) are significantly elevated in the hippocampus relative to TNF receptor 2 (TNFR2) in aged (22 months) but not young adult (6 months) Fischer 344 rats. To determine if altered TNF/TNFR1 interactions contribute to key brain aging biomarkers, aged rats received chronic (4–6 week) intracranial infusions of XPro1595: a soluble dominant negative TNF that preferentially inhibits TNFR1 signaling. Aged rats treated with XPro1595 showed improved Morris Water Maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased protein levels for the GluR1 type glutamate receptor, and lower L-type voltage sensitive Ca2+ channel (VSCC) activity in hippocampal CA1 neurons. The results suggest that diverse functional changes associated with brain aging may arise, in part, from selective alterations in TNF signaling

XPro1595 improves learning and synaptic measures. <i>A,</i>

<p>For behavioral assays, XPro1595 (0.08 mg/kg/day) was administered to the hippocampus over a six week period. Performance (mean ± SEM) in the spatial version of the Morris Water Maze was measured by path length (cm), and normalized (%) to block one performance levels for each rat. Each group showed significant improvement by block 6 (<i>p</i><0.01). However, XPro1595-, but not vehicle-treated, rats showed significant improvement as early as block 2 and 3, suggesting that TNF-blockade accelerates learning rates during aging. <b><i>B,</i></b> Mean EPSP slope amplitudes (vertical SEM bars) were plotted against mean FV amplitudes (horizontal SEM bars) to generate CA3-CA1 synaptic strength curves. Each average curve was fit with sigmoidal equations and compared across treatment groups using Z tests. Aged rats that received intraventricular infusions of XPro1595 over a four week period (0.08 mg/kg/day) exhibited a slight, but significant increase in maximal EPSP amplitude, relative to the vehicle group (<i>z</i> = 2.17). Other curve parameters (<i>i.e.</i> slope and half-maximal FV) were not affected by XPro1595. <b><i>C,</i></b> In a twin pulse paradigm (50 ms interpulse interval), the EPSP slope corresponding to pulse 2 (S2) was expressed as a percentage (mean ± SEM) of the pulse 1 (S1) EPSP slope to obtain measures of paired-pulse facilitation (PPF). No treatment effect was observed for PPF (<i>p</i> = 0.27). <b><i>D,</i></b> Time plot showing normalized mean ± SEM EPSP slope amplitudes collected before (1) and after (2) the delivery of prolonged 1 Hz stimulation (bar, 900 consecutive pulses). The inset shows representative CA1 EPSP waveforms averaged in the pre- (1) and post-1 Hz (2) periods for vehicle and XPro1595 groups. Scale bars indicate 0.5 mV vertical by 2 ms horizontal. <b><i>E,</i></b> Bar graph shows the amount of LTD, expressed as a percentage of the pre-1 Hz baseline (mean ± SEM) in each treatment condition. The results revealed significant LTD in vehicle, but not in XPro1595-treated rats (* <i>p</i><0.05, repeated measures ANOVA).</p

XPro1595 reduces L-VSCC activity in CA1 neurons of aged rats. <i>A,</i>

<p><i> Left panel</i>, Cartoon illustration of a partially dissociated hippocampal slice in which CA1 is “unzipped” along <i>stratum pyramidale</i> to expose CA1 neurons for patch clamp recording. <i>Right panel</i>, Photomicrograph showing a glass micropipette tip patched onto a CA1 pyramidal neuron in an “unzipped” slice. <b><i>B,</i></b> Three representative L-VSCC current traces and the average ensemble current (bottom trace) in cell attached patches from aged rats treated for six weeks (intrahippocampal delivery) with vehicle or XPro1595 (0.08 mg/kg/day). Traces were taken from 45–50 consecutive step depolarizations (150 ms duration) from −70 to +10 mV. <b><i>C,</i></b> Mean ± SEM current (<i>I</i>) densities (pA/μm²) for each treatment group are shown. XPro1595 significantly reduced VSCC <i>I</i> density nearly three-fold (* <i>p</i><0.05). <b><i>D–E,</i></b> Representative Western blots (<b><i>D</i></b>) and mean+SEM protein levels (<b><i>E</i></b>) for the major pore-forming L-VSCC subunits (Ca_V1.2 and Ca_V1.3) from hippocampal membrane fractions. The Na/K-ATPase served as loading control. XPro1595 did not significantly alter either Ca_V1.2 (<i>p</i> = 0.17) or Ca_V1.3 (<i>p</i> = 0.63) levels.</p

Increased TNFR1/TNFR2 ratio during aging.

<p><b><i>A,</i></b> Representative Western blots for TNFR1 and TNFR2 in hippocampal membrane fractions from young (6 mos; <i>n</i> = 6) and aged (22 mos; <i>n = </i>10) male Fischer rats, with the Na/K-ATPase loading control shown below. <b><i>B,</i></b> There was no significant change in TNFR1 protein levels with age (<i>p</i> = 0.67); however, there was a significant decrease in TNFR2 with age (* <i>p</i><0.05).</p

XPro1595 alters Iba-1 levels.

<p>XPro1595 (0.08 mg/kg/day) was administered to the hippocampus over a six week period using Alzet osmotic pumps. Immunolabeling revealed a reduction in inflammatory phenotype in microglia (<b><i>A</i></b><b>, Iba-1</b>) and, to a lesser extent, in astrocytes (<b><i>D</i></b><b>, GFAP</b>) of XPro1595 treated aged rats. CA1 = CA1 <i>stratum pyramidale</i>. Representative Western blots and mean ± SEM protein levels are for Iba-1 (<b><i>B</i></b> and <b><i>C</i></b>) and GFAP (<b><i>E</i></b> and <b><i>F</i></b>) in hippocampal fractions of vehicle and XPro1595-treated rats. β-actin and GAPDH served as loading controls for Iba-1 and GFAP, respectively. Results revealed a marked, significant reduction in Iba-1 protein levels (* <i>p</i><0.05) and a smaller, nonsignificant reduction in GFAP levels in the XPro1595 group (<i>p</i> = 0.45).</p

Effects of XPro1595 on glutamate receptor protein levels. <i>A,</i>

<p>Representative Western blots are shown for AMPA receptor (GluR1 and GluR2) and NMDA receptor subtypes (NR1, NR2A, and NR2B) in hippocampal membrane fractions from aged (22 month) rats treated for four weeks (intraventricular delivery) with vehicle or XPro1595 (0.08 mg/kg/day). The Na/K-ATPase loading control is shown below. <b><i>B,</i></b> XPro1595 treatment resulted in a selective increase in GluR1 levels (* <i>p</i><0.05).</p