Diminished trkA receptor signaling reveals cholinergic-attentional vulnerability of aging - PubMed (original) (raw)

Diminished trkA receptor signaling reveals cholinergic-attentional vulnerability of aging

Vinay Parikh et al. Eur J Neurosci. 2013 Jan.

Erratum in

Abstract

The cellular mechanisms underlying the exceptional vulnerability of the basal forebrain (BF) cholinergic neurons during pathological aging have remained elusive. Here we employed an adeno-associated viral vector-based RNA interference (AAV-RNAi) strategy to suppress the expression of tropomyosin-related kinase A (trkA) receptors by cholinergic neurons in the nucleus basalis of Meynert/substantia innominata (nMB/SI) of adult and aged rats. Suppression of trkA receptor expression impaired attentional performance selectively in aged rats. Performance correlated with trkA levels in the nMB/SI. trkA knockdown neither affected nMB/SI cholinergic cell counts nor the decrease in cholinergic cell size observed in aged rats. However, trkA suppression augmented an age-related decrease in the density of cortical cholinergic processes and attenuated the capacity of cholinergic neurons to release acetylcholine (ACh). The capacity of cortical synapses to release ACh in vivo was also lower in aged/trkA-AAV-infused rats than in aged or young controls, and it correlated with their attentional performance. Furthermore, age-related increases in cortical proNGF and p75 receptor levels interacted with the vector-induced loss of trkA receptors to shift NGF signaling toward p75-mediated suppression of the cholinergic phenotype, thereby attenuating cholinergic function and impairing attentional performance. These effects model the abnormal trophic regulation of cholinergic neurons and cognitive impairments in patients with early Alzheimer's disease. This rat model is useful for identifying the mechanisms rendering aging cholinergic neurons vulnerable as well as for studying the neuropathological mechanisms that are triggered by disrupted trophic signaling.

© 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

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Figures

Figure 1

Figure 1

In vitro screening of SIBR plasmids.

A

: Immunoblots depicting trkA expression in puromycin-selected PC12 cells transfected with UI4-SIBR-GFP plasmids expressing trkA or control shRNAs. trkA was detected as a 140 KDa band by western blot.

B

: All four shRNA constructs significantly attenuated trkA receptor expression in PC 12 cells (***p<0.001; M; SEM).

C

: Representative images of GFP-expressing, transfected PC12 cells following NGF differentiation. Compared with neurite outgrowth following transfection with the functional control (luciferase; luc) plasmid, morphometric analysis revealed marked decline in neurite length (

D

; longest neurite) and % differentiation (

E

) as a result of transfection with SIBR-trkA plasmids (all p<0.001 vs. luc) and thus impaired NGF signaling due to reduced trkA.

Figure 2

Figure 2

In vivo transduction efficiency and cholinergic targeting of AAV vectors.

A

: AAV-SIBR vector construct depicting UBC promoter-driven expression of an intronic microRNA-based shRNA (SIBR cassette) and a GFP reporter gene from the same transcript. A separate transcript expresses a puromycin resistance gene (depiction not to scale).

B

: Coronal section depicting cannula placements for AAV vector infusions for in vivo validation studies. Animals received infusions of the AAV vector expressing trkA shRNA (AAV-trkA) into the nBM/SI region of one hemisphere and of the functional control AAV vector targeting inert gene luciferase (AAV-luc) into the contralateral hemisphere. The pink-shaded clouds depict the location of major cholinergic cell groups along the medial wall of the globus pallidus (GP), that is the nucleus basalis of Meynert (nBM), ventral to the GP, that is the substantia innominata (SI) and, more ventrally, the horizontal nucleus of the diagonal band and the magnocellular preoptic region (HDB/MCPO; IC, internal capsule; CPu, caudate-putamen).

C

: GFP expressing, AAV-luc infection space in the basal forebrain, including nBM in a series of coronal sections (from Bregma −0.84 to −1.92 mm; LV: lateral ventricle). In vivo transduction efficiency of AAV vectors was determined by GFP/ChAT double-immunohistochemistry.

D

: Coronal sections depicting sampled areas in the nBM/SI region to examine colocalization of GFP and ChAT (4 weeks post-infusion). Expression of GFP column

E

) in cholinergic neurons (ChAT; column

F

) infected in vivo with a control vector four weeks post-infusion.

G

: Merged images showing colocalization of GFP and ChAT in infected BF neurons (see arrowheads for double-labelled neurons). On average, 78±6% of the cholinergic neurons in the nbM/SI region were infected.

Figure 3

Figure 3

TrkA receptor knockdown in the basal forebrain (BF) cholinergic projection system using AAV-mediated RNAi. Representative images from sections depicting trkA immunoreactivity in the nMB region of rats after 4 weeks of infusion of either the AAV-luc (

A

) or the AAV-trkA (

B

) vector (boxed areas are shown at higher magnification on right; arrows point to trkA-immunopositive neurons.

C

: AAV-trkA infusions produced a robust reduction in the cell pixel density of BF trkA-immunopositive neurons at all time points with maximum reduction in trkA levels observed 4 weeks post-AAV-trkA infusion (see Results).

D

: TrkA receptor densities in homogenates prepared from prefrontal tissue were determined using immunoblotting. Immunoblot analysis reveal a robust decline in cortical trkA expression in BF trkA knockdown animals. (Unpaired t tests: **,***p<0.01, 0.001 vs. AAV-luc).

Figure 4

Figure 4

Effects of nBM/SI trkA knockdown on attentional performance of young and aged animals.

A

: Hits to longest signals during the initial ~4 weeks post-infusion. This measure began to decline in aged rats during the third week post-infusion.

B

: The effects of the vector and age interacted to impair the animals' performance in signal trials (see Results for main statistical findings) but not in non-signal trials (

D

). Multiple comparisons are based on main effects and a significant interaction between the effects of vector, age, and signal duration (LSD: *, ** p<0.05, 0.001).

C

: Post hoc one-way ANOVA of the effects of group confirmed the selective impairment in aged/AAV-trkA rats.

E

: Omission remained low (<8%) and did not differ between conditions.

F

: Correlations between SAT scores and nMB/SI trkA density indicated a significant positive correlation in aged but not young rats (Results). Data points from animals infused with AAV-trkA are all within the circled area, reflecting a complete separation of trkA levels as a function of vector. Additional analysis also indicated that, trkA levels in (young and aged) control animals generally correlated with the animals hit rate; infusions of AAV-trkA abolished this relationship.

Figure 5

Figure 5

Depolarization-evoked ACh release in prefrontal cortex.

A

: Representative traces of choline signals recorded amperometrically using choline-sensitive microelectrodes placed in the prelimbic regions of the medial PFC. Release was evoked by pressure ejections of KCl via co-implanted micropipettes. These traces illustrate the attenuated amplitude of evoked ACh release in aged/AAV-trkA rats.

B

: A significant interaction between the effects of age and vector (Results) reflect the greater AAV-trkA-induced attenuation of ACh release capacity in aged animals when compared with young animals (LSD; *p<0.05; **p<0.001).

Figure 6

Figure 6

Cholinergic neurons in the nMB/SI and cortical cholinergic processes in young and aged animals after AAV-mediated trkA knockdown. Sections were processed for ChAT immunohistochemistry.

A

: Representative images from coronal sections depicting ChAT-immunopositive neurons in the nBM/SI region. The inset shows examples of cholinergic neurons at a higher magnification. As described in Results, neither age nor vector affected cholinergic cell counts.

B

: Cholinergic cell area was reduced in aged animals as compared to young animals; however, this measure was not affected by vector and the effects of age and vector did not interact.

C

: ChAT levels expressed as % ChAT-positive pixels in the analyzed area (see Methods). There was no discernible difference in ChAT levels between young AAV-luc and AAV-trkA animals. Multiple comparisons were based on significant effects of and interaction between age and vector. ChAT levels declined with age and were lowest in aged/AAV-trkA rats.

D,E

: Paralleling ChAT levels in the nMB/SI region, the density of ChAT-positive puncta in the prefrontal cortex was significantly lower in aged animals in general, and lower in aged/AAV-trkA rats when compared with aged control (aged/AAV-luc) and young/AAV-trkA rats. The counting area is symbolized in the coronal schematic on the left. Note that dark-stained cholinergic, bipolar interneurons were not included into cortical counts (LSD: *, **, p<0.05, 0.01).

Figure 7

Figure 7

Effects of age and trkA knockdown on cortical NGF/proNGF levels and cortical and basal forebrain NGF receptor densities. NGF levels were estimated in cortical homogenates using ELISA. Neither age nor infusion of the vector affected cortical NGF levels (not shown).

A

: In contrast, proNGF levels were higher in aged rats and in rats infused with AAV-trkA vector. Representative immunoblots of cortical proNGF from duplicate samples are shown on top. proNGF immunoreactive band (26 KDa) was detected using a rabbit monoclonal anti-proNGF antibody. Bar graphs (bottom) show proNGF densities normalized to β-tubulin.

B

: Cortical p75 receptor density was increased in aged animals but remained unaffected by infusion of the vector. Blots depict a 75 KDa band corresponding to p75 receptor.

C

: Cortical trkA receptor density was unaffected by age but, as expected, decreased as a result of infusion of the AAV-trkA vector.

D,E

: trkA receptor immunopositive neurons in the nMB/SI region of young rats infused with either the control or the AAV-trkA vector (left, right in

D

). Similar to cortical trkA levels, nMBI/SI levels were unaffected by age but robustly suppressed by the AAV-trkA vector (

E

).

F,G

: p75 receptor expression in the nMB/SI region was neither affected by age nor by infusion of the AAV-trkA vector (see Discussion for the contrasting age-related increase in cortical p75 levels; see Results for ANOVAs; post hoc comparisons: *, p<0.05; **, p<0.001).

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