ProNGF Drives Localized and Cell Selective Parvalbumin Interneuron and Perineuronal Net Depletion in the Dentate Gyrus of Transgenic Mice - PubMed (original) (raw)
ProNGF Drives Localized and Cell Selective Parvalbumin Interneuron and Perineuronal Net Depletion in the Dentate Gyrus of Transgenic Mice
Luisa Fasulo et al. Front Mol Neurosci. 2017.
Abstract
ProNGF, the precursor of mature Nerve Growth Factor (NGF), is the most abundant NGF form in the brain and increases markedly in the cortex in Alzheimer's Disease (AD), relative to mature NGF. A large body of evidence shows that the actions of ProNGF and mature NGF are often conflicting, depending on the receptors expressed in target cells. TgproNGF#3 mice, expressing furin-cleavage resistant proNGF in CNS neurons, directly reveal consequences of increased proNGF levels on brain homeostasis. Their phenotype clearly indicates that proNGF can be a driver of neurodegeneration, including severe learning and memory behavioral deficits, cholinergic deficits, and diffuse immunoreactivity for A-beta and A-beta-oligomers. In aged TgproNGF#3 mice spontaneous epileptic-like events are detected in entorhinal cortex-hippocampal slices, suggesting occurrence of excitatory/inhibitory (E/I) imbalance. In this paper, we investigate the molecular events linking increased proNGF levels to the epileptiform activity detected in hippocampal slices. The occurrence of spontaneous epileptiform discharges in the hippocampal network in TgproNGF#3 mice suggests an impaired inhibitory interneuron homeostasis. In the present study, we detect the onset of hippocampal epileptiform events at 1-month of age. Later, we observe a regional- and cellular-selective Parvalbumin interneuron and perineuronal net (PNN) depletion in the dentate gyrus (DG), but not in other hippocampal regions of TgproNGF#3 mice. These results demonstrate that, in the hippocampus, the DG is selectively vulnerable to altered proNGF/NGF signaling. Parvalbumin interneuron depletion is also observed in the amygdala, a region strongly connected to the hippocampus and likewise receiving cholinergic afferences. Transcriptome analysis of TgproNGF#3 hippocampus reveals a proNGF signature with broad down-regulation of transcription. The most affected mRNAs modulated at early times belong to synaptic transmission and plasticity and extracellular matrix (ECM) gene families. Moreover, alterations in the expression of selected BDNF splice variants were observed. Our results provide further mechanistic insights into the vicious negative cycle linking proNGF and neurodegeneration, confirming the regulation of E/I homeostasis as a crucial mediating mechanism.
Keywords: E/I imbalance; dentate gyrus; expression profile; extracellular matrix; interneurons; parvalbumin; proNGF; transgenic mice.
Figures
Figure 1
(A–D) Parvalbumin+ interneuron depletion in the DG of TgproNGF#3 mice. (A–C) Quantification of Parvalbumin+ interneurons (Parv+ interneuron density, expressed as cells/mm3) in the DG of wt and TgproNGF#3 mice (3, 6, and 12 ms-old). Cell counting was performed on at least 4–6 sections per animal, males and females (n = 4 per each animal group). Bars and lines are representative of mean ± SEM (_T_-test, **P < 0.005; *P < 0.05). (D) Confocal micrographs of Parvalbumin immunofluorescence in 12-month-old wt and TgproNGF#3 mice hippocampus (DG region). Scale bar: 75 micron. (E,F) Parvalbumin+ interneuron depletion in the amygdala of 12-month-old TgproNGF#3 mice. (E) Quantification of Parv+ interneurons in the basolateral amygdala of 6 and 12-month-old wt and TgproNGF#3 mice (Parv+ interneuron density, expressed as cells/mm3). Cell counting was performed on the lateral region of the amygdala on both sides on at least 4–6 sections per animal, males and females (n = 4 per each animal group) (_T_-test, *P < 0.05). (F) Confocal micrographs of Parvalbumin immunofluorescence labeling in the amygdala of 12-month-old wt and TgproNGF#3 mice (Parv+ neurons are located in the lateral region of the amygdala). Scale bar: 75 micron.
Figure 2
Double labeling for Parvalbumin and GAD65-67 in the dentate gyrus (DG) of 12-months-old wt and TgproNGF#3 mice. (A) Quantification of GAD+/Parv+ neurons of wt and TgproNGF#3 mice (3- and 12-month old), expressed as percentage. Cell counting was performed on the DG on both sides, on at least 4–6 sections per animal, males and females (n = 4 per each animal group) (_T_-test, **P < 0.005). (B) Confocal Micrographs of Parv/GAD65-67 double immunofluorescence labeling in the DG of wt and TgproNGF#3 mice. Reduction of both Parv and GAD markers was observed. Scale bar: 50 micron.
Figure 3
Calbindin immunoreactivity is unaffected in 3-month-old TgproNGF#3 mice, but markedly reduced in the DG and CA3 region of the hippocampus of 12-month-old TgproNGF#3 mice. (A) Quantification of fluorescence intensity for calbindin (expressed as Corrected Integrated Density/mm2) in the DG region of 3-month-old wt and TgproNGF#3 mice. (B) Confocal micrographs of calbindin immunofluorescence labeling in 3-month-old wt and TgproNGF#3 mice (DG region of the hippocampus). Scale bar: 50 micron. (C) Quantification of fluorescence intensity for calbindin (expressed as Corrected Integrated Density/mm2) in the DG region of 12-month-old wt and TgproNGF#3 mice (_T_-test, *P = 0.01). (D) Quantification of fluorescence intensity for calbindin (expressed as Corrected Integrated Density/mm2) in the CA3 region of 12-month-old wt and TgproNGF#3 mice (_T_-test, *P = 0.01). Analysis was performed on at least 4–6 sections per animal, males and females (n = 4 per each animal group). (E) Reduced Calbindin immunostaining in DG region of the hippocampus (upper panels) and in CB+ axons (Mossy fibers) projecting to the CA3 region (lower panels) in 12-month-old wt and TgproNGF#3 mice. (E, upper panels) Confocal micrographs of calbindin immunofluorescence labeling in 12-month-old wt and TgproNGF#3 mice in DG region of the hippocampus. Scale bar: 25 micron. (E, lower panels) Confocal micrographs of calbindin immunofluorescence labeling in 12-month-old wt and TgproNGF#3 mice in CA3 region in 12-month-old wt and TgproNGF#3 mice. Scale bar: 50 micron.
Figure 4
(A,B) The perineuronal nets (PNN) are markedly reduced in the dentate gyrus of TgproNGF#3 mice. Confocal microscopy. (A) Quantification of Parvalbumin+ interneurons of wt and TgproNGF#3 mice (3-, 6-, and 12-month-old). % total Parv+ neurons − blue bar − and Parv+ neurons ensheathed by the PNN − red bar. The reduction in Parv+ neurons ensheathed by PNN is significant in 6- and 12-month TgproNGF#3 mice. Analysis was performed on at least 4–6 sections per animal, males and females (n = 4 per each animal group) (_T_-test, *P < 0.005). (B) Double immunofluorescence for the perineuronal net and for Parvalbumin in the DG of 12-months-old wt and TgproNGF#3 mice. Brain sections were labeled with anti-Parvalbumin antibody, with the lectin Wisteria Floribunda (an agglutinin commonly used to map PNN and ECM, since it binds the glycan component of PNN proteoglycans) and Dapi. Scale bar: 50 micron. (C) Double immunofluorescence for aggrecan (a component of the PNN surrounding Parv+ interneurons) and for Parvalbumin in the DG of 12-months old wt and TgproNGF#3 mice. Scale bar: 50 micron.
Figure 5
(A) Overall statistics of differentially expressed mRNAs in the hippocampus of 1-, 3-, and 12-months old TgproNGF#3 mice. Only transcripts with Limma p < 0.05 and |Log2 fold change| > 1.0 were selected. The graph shows the overall down-regulation of transcripts. (B,C) Validation of selected genes by qRT-PCR, respectively, at 3- and 12-months of age. The Log2 fold change (Log2 FC) ratio in microarray (red) and qRT-PCR (blue) are shown. The error bar is the S.E.M. and (*) indicates statistical significance by _T_-test (*P < 0.05).
Figure 6
Functional analysis of specific gene categories. The whole dataset was analyzed by the tool GSEA (Gene Set Enrichment Analysis). FDR q < 0.05 (statistically significant) is indicated by (*) (obtained by gene set permutation). The GSEA analytical tool allows to identify small changes affecting a large set of functionally related genes compared to a per gene statistics. GSEA analytical tool allows to identify with high sensitivity global up- or down-regulation trends affecting large sets of functionally related genes. GSEA plots represent, for each functional category, a kind of summation integral of expression change for all single genes in the category. The methodology is sensitive even for small changes involving large gene sets. The GSEA plots highlight a general down-regulation of the Long Term Potentiation and Transcription systems at 3-months, while the Collagen and Extracellular Matrix categories are up-regulated at 3-months and down-regulated at 12-months. In each panel, the green line is the GSEA score plot, while the gray plot on the bottom of each panel is the global distribution of Log2 fold change for all genes, with up-regulated genes on the left and down-regulated ones on the right. The black thin bars on the bottom of each sub-panel indicate the genes belonging to the functional category, with each bar position corresponding to the specific score and Log2 fold change levels in the two plots. The following link provides information about how to interpret GSEA data and plot:
http://software.broadinstitute.org/gsea/doc/GSEAUserGuideFrame.html?Interpreting\_GSEA
.
Figure 7
(A) Expression level of Bdnf gene isoforms, by qRT-PCR, at 1-month of age. (a) Differential expression of main Bdnf isoforms, measured ad Log2 fold change ratio TgproNGF#3 vs. Wild Type; (*) indicates a statistical significant variation by 1-tail _T_-test for Bdnf1, while the two rightmost bars indicates the global relative expression of short and long isoforms. (b) Relative distribution of short and long Bdnf forms (left) and of the different isoforms (right) in the TgproNGF#3 and Wild Type mice: the relative proportion of short and long isoforms is comparable in the two mouse strains. (B) Expression level of Bdnf gene isoforms, by qRT-PCR, at 3-months of age. (a) Differential expression of main Bdnf isoforms, measured ad Log2 fold change ratio TgproNGF#3 vs. Wild Type; (*) indicates a statistical significant variation by 1-tail _T_-test, for Bdnf3 and Bdnf5, while the two rightmost bars indicates the global relative expression of short and long isoforms. (b) Relative distribution of short and long Bdnf forms (left) and of the different isoforms (right) in the TgproNGF and Wild Type mice: the relative proportion of short and long isoforms is comparable in the two mouse strains. (C) Expression level of Bdnf gene isoforms, by qRT-PCR, at 12-months of age. (a) Differential expression of main Bdnf isoforms, measured ad Log2 fold change ratio TgproNGF#3 vs. Wild Type; (*) indicates a statistical significant variation by 1-tail _T_-test, for Bdnf1 and Bdnf2B, while the two rightmost bars indicates the global relative expression of short and long isoforms. (b) Relative distribution of short and long Bdnf forms (left) and of the different isoforms (right) in the TgproNGF#3 and Wild Type mice: the relative proportion of short and long isoforms is comparable in the two mouse strains.
Figure 8
Age-dependent modifications of epileptiform activity in TgproNGF#3 mice. (A–C left panels) Photographs of EC-HP slices obtained from a 1-, 3-, and 12-months old TgproNGF#3 mouse, respectively, placed over an array of 64 planar multi-electrodes, detectable in transparency through the slice. Colored dots indicate position of the electrodes, whose voltage signals appear in the corresponding colored area. (A–C right panels) Simultaneous recordings from each one of the 64 electrodes. (A–C insets) Trace records from the HP (red) and EC (green) detected by the colored electrodes in the corresponding photograph, showing no spontaneous activity in the EC of 1- and 3-months old mice, as opposed to spontaneous cortical events at 12-month, while the hippocampal area displays spontaneous events at all 3 ages.
Figure 9
Schematic representation of hypothesized targets of proNGF/NGF imbalance. (A) We hypothesize that proNGF/NGF imbalance primarily impacts Parv+ interneurons that make inhibitory synaptic contact with DG granule cells that project their axons (the so-called Mossy fibers) to CA3 neurons. These interneurons normally produce NGF and feed cholinergic fibers deriving from NGF-dependent BFN expressing p75NTR, making synaptic contact with Parv interneuron dendrites in the DG. In such a way, cholinergic input tunes Parv+ interneuron inhibitory output onto granule cells, affecting their excitatory activity to CA3 neurons (mediated by Mossy fibers). (B) Overexpression of furin-resistant proNGF, producing an NGF/proNGF imbalance, severely impairs cholinergic fibers, disrupting this feed-back control mechanism influencing hippocampal memory encoding and retrieval and contributing to E/I imbalance. I, interneuron; Parv+, parvalbuminergic interneuron; GC, granule cell; DG, dentate gyrus; BFN, Basal Forebrain Neuron.
References
- Baj G., Del Turco D., Schlaudraff J., Torelli L., Deller T., Tongiorgi E. (2013). Regulation of the spatial code for BDNF mRNA isoforms in the rat hippocampus following pilocarpine-treatment: a systematic analysis using laser microdissection and quantitative real-time PCR. Hippocampus 23, 413–423. 10.1002/hipo.22100 -DOI -PubMed
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