Silencing Alpha Synuclein in Mature Nigral Neurons Results in Rapid Neuroinflammation and Subsequent Toxicity

Abstract

Human studies and preclinical models of Parkinson's disease implicate the involvement of both the innate and adaptive immune systems in disease progression. Further, pro-inflammatory markers are highly enriched near neurons containing pathological forms of alpha synuclein (α-syn), and α-syn overexpression recapitulates neuroinflammatory changes in models of Parkinson's disease. These data suggest that α-syn may initiate a pathological inflammatory response, however the mechanism by which α-syn initiates neuroinflammation is poorly understood. Silencing endogenous α-syn results in a similar pattern of nigral degeneration observed following α-syn overexpression. Here we aimed to test the hypothesis that loss of α-syn function within nigrostriatal neurons results in neuronal dysfunction, which subsequently stimulates neuroinflammation. Adeno-associated virus (AAV) expressing an short hairpin RNA (shRNA) targeting endogenous α-syn was unilaterally injected into the substantia nigra pars compacta (SNc) of adult rats, after which nigrostriatal pathology and indices of neuroinflammation were examined at 7, 10, 14 and 21 days post-surgery. Removing endogenous α-syn from nigrostriatal neurons resulted in a rapid up-regulation of the major histocompatibility complex class 1 (MHC-1) within transduced nigral neurons. Nigral MHC-1 expression occurred prior to any overt cell death and coincided with the recruitment of reactive microglia and T-cells to affected neurons. Following the induction of neuroinflammation, α-syn knockdown resulted in a 50% loss of nigrostriatal neurons in the SNc and a corresponding loss of nigrostriatal terminals and dopamine (DA) concentrations within the striatum. Expression of a control shRNA did not elicit any pathological changes. Silencing α-syn within glutamatergic neurons of the cerebellum did not elicit inflammation or cell death, suggesting that toxicity initiated by α-syn silencing is specific to DA neurons. These data provide evidence that loss of α-syn function within nigrostriatal neurons initiates a neuronal-mediated neuroinflammatory cascade, involving both the innate and adaptive immune systems, which ultimately results in the death of affected neurons.

Keywords: alpha-synuclein; knockdown; major histocompatibility complex class 1 (MHC-1); microglia; neuroinflammation.

Figure 1

The time course and concentration response of tyrosine hydroxylase positive (TH+) cell loss following α-syn knockdown within nigrostriatal neurons. Rats received unilateral substantia nigra pars compacta (SNc) injections of AAV2/5 expressing an α-syn short hairpin RNA (shRNA) or a myocardin (Myo) control shRNA. AAV2/5 expressing the respective shRNAs was injected at a titer of 2.6 × 10¹² vg/ml (Full) or 1.3 × 10¹² vg/ml (Half). Rats were then sacrificed at 7, 10, 14, and 21 days post-surgery, and the number of TH+ neurons in the SNc was estimated using unbiased stereology. Columns in panel (A) represent mean number of TH+ cells in the injected SNc, expressed as the percent of TH+ cells in the contralateral SNc, +1 SEM (n = 6–7/group), in animals receiving Myo Full shRNA (solid blue columns), α-syn Full shRNA (solid red columns), Myo Half shRNA (hatched blue columns), or α-syn Half shRNA (hatched red columns). *Significantly different (ANOVA: p < 0.05). Representative images of TH positive neurons of the SNc for all shRNA concentrations and time points are shown panels (B–Q). Arrowheads indicate areas in which TH+ cell loss was observed. Scale bar in (Q) represents 500 μm and applies to all other panels.

Figure 2

Time course and concentration response of nigrostriatal axon terminal degeneration following α-syn knockdown. Rats received unilateral injections of AAV2/5 expressing the α-syn shRNA or a myocardin control shRNA. AAV2/5 expressing the respective shRNAs was injected at a titer of 2.6 × 10¹² vg/ml (Full) or 1.3 × 10¹² vg/ml (Half). Rats were then sacrificed at 7, 10, 14 and 21 days post-surgery, and striatal TH, VMAT, dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were quantified. Columns represent the mean total signal intensity of striatal TH (A) or VMAT (R), or the mean concentration of striatal DA (II), DOPC (JJ) or the DOPAC to DA ratio (KK), expressed as the percent of the contralateral striatum, +1 SEM (n = 5–8/group), in animals receiving Myo Full shRNA (solid blue columns), α-syn Full shRNA (solid red columns), Myo Half shRNA (hatched blue columns), or α-syn Half shRNA (hatched red columns). *Significantly different (ANOVA, p < 0.05). Representative images of TH+ (B–Q) and VMAT+ (S–HH) striata are shown. Arrowheads in panels (I,Q,Z) indicate areas in which nigrostriatal terminal loss was observed.

Figure 3

α-syn knockdown increases reactive microglia in the SNc. Rats received a single injection of AAV2/5 (2.6 × 10¹² vg/ml; Full) expressing a target α-syn shRNA or a myocardin control shRNA. Rats were then sacrificed at 7, 10, 14 and 21 days post-surgery, the number of IBA1+ microglia were quantified using unbiased stereology. Columns in panel (A,B) represent mean number of IBA1+ microglia in the ipsilateral SNc and substantia nigra pars reticulata (SNr), respectively, expressed as the percent of IBA1+ microglia in the contralateral SNc and SNr, +1SEM (n = 5–8/group), in animals receiving Myo Full shRNA (solid blue columns), α-syn Full shRNA (solid red columns). Representative images show low and high magnification images of IBA1+ microglia within the SNc and SNr in animals treated with the α-syn shRNA (C–F) or myocardin shRNA (G–J). Panel (K) depicts CD68+ (red), IBA1+ (teal) microglia within the anatomical boundaries of the SNc 10-days following injection of the α-syn shRNA. Arrowheads in panels (L–N) depict CD68+ (red), IBA1+ (teal) reactive microglia surrounding α-syn shRNA transduced neurons (indicated by green fluorescent protein (GFP) reporter; green) at 10- (L), 14- (M), and 21-days (N) post-injection. Panels (O–R) depicts IBA1+ microglia that are largely devoid of CD68 immunoreactivity within the anatomical boundaries of the SNc at 10 (O,P), 14 (Q) and 21 days (R) following injection of α-myocardin shRNA. Numerous round CD68+ cells, which expressed little to no IBA1, were found throughout the entire ventral midbrain 21-days post-α-syn-shRNA expression (S,T). Panel (U) corresponds to area within the box in panels (S,T). The yellow arrow in panels (V,W) depicts the normal punctate CD68 immunoreactivity that was observed within IBA1+ microglia. The white arrowheads in panels (V,W) depict ameboid cells expressing high levels of CD68 with little to no colocalization with IBA1. *Significantly different (ANOVA: P < 0.05). The scale bars in panel (J) and the inset in panel (J) represents 1 mm and 100 μm respectively, and apply to the corresponding panels and insets in (C–I). The scale bar in panel (O) represents 50 μm and applies to panel (K). The scale bar in panel (R) represents 25 μm and apply to panels (L,M,P,Q). Scale bar in panel (T) represents 250 μm and applies to panel (S). Scale bar in panels (U,V) represent 25 μm. Scale bar in Panel (W) represents 10 μm.

Figure 4

Silencing α-syn in nigral neurons induces major histocompatibility complex class 1 (MHC-1) expression. Rats received a single injection of AAV2/5 (2.6 × 10¹² vg/ml; Full) expressing a target α-syn shRNA or a myocardin control shRNA. Rats were then sacrificed at 10, 14, and 21 days post-surgery. Panels (A–X) show MHC-1 (red) immunoreactivity near TH (green) neurons in myocardin shRNA treated animals at 10 days (A–D), 14 days (I–L) and 21 days (Q–T) post injections. MHC-1 expression in α-syn shRNA treated animals at 10 days (E–H), 14 days (M–P) and 21 days (U–X) post injection. White arrowheads in (F–H) and (N–P) indicate neuronal MHC-1 expression. Panels (Y–CC) show co-localization of MHC-1 (red) with the neuronal marker HUc (teal) within the SNc 10 days following α-syn shRNA injection. Panels (DD–JJ) show co-localization of MHC-1 expression in TH+ neurons of the α-syn shRNA injected SNc. White arrowheads in (DD–FF) indicate MHC-1 expression in a neuron that also expresses low levels of TH. Yellow arrows in (GG–II) indicate MHC-1 expressing microglia. Panel (JJ) shows a composite z-stack image acquired using confocal microscopy, corresponding to the area within the box in panels (GG–II) showing MHC-1 expression on the surface of a TH+ neuron. Panels (KK–TT) show representative micrographs of transduced neurons (GFP+; green) within the α-syn shRNA injected SNc stained for IBA1 (teal) and MHC-1 (red). White arrowheads in (KK–NN) indicate cell surface expression of MHC-1 on GFP+ transduced neurons, which does not co-localize with the microglial marker IBA1 (OO). Yellow arrows in (TT) indicate IBA1+ microglia surrounding a neuron with high MHC-1 expression. Panels (QQ–TT) correspond to the area within the box in (PP). Panels (OO) and (TT) are composite z-stack images acquired using confocal microscopy. Columns in (UU) represent the mean number of MHC-1+ neurons, + 1 SEM (n = 4–5/group), counted from four sections of the ipsilateral ventral midbrain, spanning the rostral-caudal axis of the SNc. Columns in (VV) represent the mean MHC-1 fluorescent intensity within ventral midbrain of the ipsilateral hemisphere, expressed as a percent of the mean MHC-1 fluorescent intensity within ventral midbrain of contralateral hemisphere, +1 SEM (n = 4–5/group). *Significantly different (ANOVA: P < 0.05). Scale bar in (U) represents 500 μm and applies to panels (A,E,I,M,Q). Scale bar in (X) represents 100 μm and applies to (B–D,F–H,J–L,N–P,R–T,V,W). Scale bar in panel (BB) represents 10 μm and applies to panels (Y–AA). Scale bar in (II) represents 25 μm and applies to panels (DD–HH). Scale bars in (NN) and (SS) represent 25 μm and apply to panels (KK–MM,PP–RR).

Figure 5

Silencing α-syn in nigral neurons results in the infiltration of CD3+ T-cells to the SNc. Rats received a single injection of AAV2/5 (2.6 × 10¹² vg/ml; Full) expressing the α-syn or myocardin shRNA and were sacrificed 21 days post-surgery. Panels show CD3+ (red) T-cells near TH+ (green) nigral neurons in the SNc of α-syn shRNA (A–D) and myocardin shRNA (E–H) treated animals. White arrowheads in panels (B–D) indicate CD3+ T-cells. Scale bar in panel (F) represents 250 μm and applies to panel (A,B,E). Scale bar in panel (G) represents 50 μm and applies to panel (C). Scale bars in panels (H) represents 10 μm and applies to panel (D).

Figure 6

The effects of α-syn knockdown within purkinje cells of the cerebellum. Rats received a single injection of AAV2/5 (2.6 × 10¹² vg/ml; Full) expressing an α-syn shRNA or a myocardin control shRNA. One-month post-surgery rats were sacrificed. Panels (A,B) show transduction within the granule cell layer (GCL) of the cerebellum, directly adjacent to the purkinje cell layer (PCL). High magnification of the area within the box in panel (A) is shown in panel (B). Panels (C–H) shows IBA1+ microglia in Myo (C–E) and α-syn shRNA (F–H) treated animals. Unbiased stereology was used to quantify NeuN+ and calbindin+ neurons in the cerebellum. Columns in Panel (M) represent the mean number of NeuN+ cells from animals treated with the myocardin shRNA (black bars) or the α-syn shRNA (white bars). Representative images of NeuN+ cells of the cerebellum are shown for myocardin shRNA (I,J) and α-syn shRNA (K,L) treated animals. Columns in (R) represent mean number of calbindin+ cells within the cerebellum from animals injected with the myocardin shRNA (black bars) or the α-syn shRNA (white bars). Representative images of calbindin immunoreactivity within purkinje cells of the cerebellum from animals injected with the myocardin shRNA (N,O) and α-syn shRNA (P,Q) are shown. Error bars in (M,R) are + 1 SEM (n = 7–8/group). *Significantly different than myocardin treated group (t-test: P < 0.05). Panels (S–Z) show co-localization of calbindin (teal) with the pan-neuronal marker HUc (red) in the PCL of animals treated with the myocardin shRNA (S–V) or the α-syn shRNA (W–Z). High magnification images of the boxes in panels (S,W) are shown in panels (T–V) and (X–Z), respectively. Arrows indicate purkinje cells that are calbindin positive (T–V) or calbindin negative (X–Z). Columns in panel (AA) represent the mean number of purkinje cells in the third lobule of the cerebellar vermis (3Cb) from animals treated with the myocardin shRNA (black bars) or the α-syn shRNA (white bars). Scale bars in panels (A,B) represent 250 μm and 50 μm respectively. Scale bar in panels (G) represents 50 μm and applies to panel (D). Scale bar in panel (H) represents 25 μm and applies to panel (E). Scale bar in panel (Q) represents 100 μm and applies to panels (J,L,O). Scale bar in the inset in panel (Q) represents 25 μm and applies to the inset in panel (O). Scale bar in panel (W) represents 100 μm and applies to panel (S). Scale bar in panel (Z) represents 25 μm and applies to panels (T–V) and (X,Y).

Figure 7

A proposed mechanism of α-syn shRNA induced toxicity. Healthy, TH+ (green) nigral neurons express high levels of α-syn (pink dots) (A). Delivery of adeno-associated virus (AAV) expressing an shRNA targeting endogenous α-syn results in a rapid knockdown of α-syn expression that was detected as early as 7 days post-delivery (B). Silencing endogenous α-syn results in a progressive decrease in TH expression within individual nigral neurons, as well as an overall loss of TH+ neurons within the SNc. Coinciding with loss of TH expression, nigral neurons up-regulate cell surface expression of MHC-1 as early as 10 days post-injection (C). MHC-1 up-regulation results in recruitment of reactive microglia and the infiltration of CD3+ T-cells to the injected SNc. Recruited immune cell then surround and engulf MHC-1+ neurons, culminating in the death of nigral neurons via the induction of apoptotic cell death (red nucleus), and a corresponding loss of nigrostriatal terminals (D,E). Cytokines released from reactive microglia in the affected SNc cause neighboring, non-transduced nigral neurons to increase surface expression of MHC-1, creating a cycle of toxicity (E).