Dendritic spinopathy in transgenic mice expressing ALS/dementia-linked mutant UBQLN2

Abstract

Mutations in the gene encoding ubiquilin2 (UBQLN2) cause amyotrophic lateral sclerosis (ALS), frontotemporal type of dementia, or both. However, the molecular mechanisms are unknown. Here, we show that ALS/dementia-linked UBQLN2(P497H) transgenic mice develop neuronal pathology with ubiquilin2/ubiquitin/p62-positive inclusions in the brain, especially in the hippocampus, recapitulating several key pathological features of dementia observed in human patients with UBQLN2 mutations. A major feature of the ubiquilin2-related pathology in these mice, and reminiscent of human disease, is a dendritic spinopathy with protein aggregation in the dendritic spines and an associated decrease in dendritic spine density and synaptic dysfunction. Finally, we show that the protein inclusions in the dendritic spines are composed of several components of the proteasome machinery, including Ub(G76V)-GFP, a representative ubiquitinated protein substrate that is accumulated in the transgenic mice. Our data, therefore, directly link impaired protein degradation to inclusion formation that is associated with synaptic dysfunction and cognitive deficits. These data imply a convergent molecular pathway involving synaptic protein recycling that may also be involved in other neurodegenerative disorders, with implications for development of widely applicable rational therapeutics.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Cognitive deficits in UBQLN2P497H mice. (A) Spatial learning and memory of UBQLN2P497H and nontransgenic (NT) control mice (11–13 mo) was assessed by spontaneous alternation in the Y maze (n = 4–7 per group). UBQLN2P497H mice exhibited random arm selection (52%) instead of the spontaneous alternation of control mice (P = 0.09). (B) The total number of arm entries was higher for UBQLN2P497H mice, indicating that they do not have a motor deficit. (C and D) Delay fear conditioning was tested by assessing freezing behavior to the same context (C) and to the conditioned stimulus (CS) in a novel context (D) 24 h after training. The average percent freezing is shown as an index of delay fear memory (n = 5–7 per group). Note that the UBQLN2P497H mice showed significantly lower levels of freezing than control mice in delay fear conditioning. (E and F) Mice were trained with six trials (three blocks of two trials) per day for 5 d in the Morris water maze. The average latency and path length to reach the hidden platform is plotted across training days (n = 7–11 per group). ANOVA indicated a significantly shorter escape latency for the NT mice (F1,16 = 7.2, P = 0.02), and a decrease in escape latency across sessions (F4,64 = 9.2, P < 0.0001). ANOVA also indicated a significantly shorter path length for the NT mice (F1,16 = 7.4, P = 0.01), and a decrease in path length across sessions (F4,64 = 15.6, P < 0.0001). Each data point represents the mean ± SEM. The longer escape latency and longer path length of the UBQLN2P497H mice is indicative of cognitive impairment. *P < 0.01, **P < 0.0001 versus NT compared by ANOVA and post hoc Fisher's protected least significant difference (PLSD) test.

Fig. 2.

Ubiquilin2 pathology in transgenic mice. Immunohistochemistry with the ubiquilin2-N antibody (2) was performed using brain sections from UBQLN2P497H transgenic mice of different ages. (A) An overall image shows ubiquilin2-positive aggregates in the hippocampus of a 4-mo-old mouse. (B–J) Representative ubiquilin2-positive aggregates in the dentate gyrus (B, E, and H), CA3 (C, F, and I), and CA1 (D, G, and J) from mice of 2 mo (B–D), 3 mo (E–G), and 15 mo (H–J) are shown. Ubiquilin2 pathology is shown in transgenic, but not nontransgenic mice (Inset), even though diffused cytosolic ubiquilin2 reactivity is well shown in some neuronal cells, especially in the pyramidal neurons. (Scale bars: A, 200 µm; B–J, 50 µm.)

Fig. 3.

Ubiquilin2 pathology in other regions of the brain. Immunohistochemistry with a ubiquilin2 antibody (ubiquilin2-N) was performed on the brain sections from 15-mo-old UBQLN2P497H transgenic (TG), and age- and sex-matched nontransgenic mice (NT). Representative images from subiculum (A), entorhinal cortex (B), neocortex (C), cerebellum (D), putamen (G), and corpus callosum (H) of TG mice are indicated. Representative images from subiculum (E) and cerebellum (F) of NT mice are shown for comparison. Ubiquilin2 aggregates are predominantly present in the gray matter (A–D), but not white matter (D, G, and H). (Scale bars: A–F, 50 µm; G and H, 100 µm.)

Fig. 4.

Protein aggregates in dendritic spines and stems. (A–D) Electron microscopy was performed using the hippocampus and the frontal cortex of a 15-mo-old UBQLN2P497H transgenic mouse. Representative protein aggregates containing membrane-free and flocculent-to-granulofibrillar material are illustrated in the dendritic spines and stems (A–D). (A) The inclusion (asterisk) occupies a large area of an obliquely sectioned, midsized dendritic trunk (indicated by D). Some filamentous elements within the inclusion are indicated by arrows; axodendritic synapses are indicated by arrowheads. The fine structure of mitochondria (unlabeled) appears unaltered. (B) A large granulofibrillar inclusion (asterisk) fills the ballooning portion of a small stem dendrite (indicated by D). Two enlarged spines (ES) containing flocculent material emanate from the dendritic trunk (arrows) and a normal spine (arrowhead) from the ballooned portion. (C) A small dendritic trunk (indicated by D) contains organelles with unchanged fine structure and emits a swollen, thin-necked spine (ES) filled by granulofibrillar material. Three spines with normal fine structure are labeled (S). Split postsynaptic density is indicated by arrowheads. A hypertrophic spine apparatus is indicated by an arrow. (D) A small dendritic trunk (D1) emits two spines (S) and has apparently normal ultrastructure, whereas another small dendrite (D2) has an enlarged spine (ES) with granulofibrillar contents. A spine apparatus is indicated by an arrow and synaptic junctions by arrowheads. (Scale bars: 0.5 µm.)

Fig. 5.

Altered synaptic plasticity in the UBQLN2P497H mice. Representative images of the molecular layer of dentate gyrus of 15-mo-old control (A and C) and UBQLN2 transgenic mice (B and D). The red arrows in A and B indicate the areas magnified to display the spines presented in C and D. GL, granule cell layer of dentate gyrus; ML, molecular layer of dentate gyrus. (Scale bar in B, 50 µm, also applies to A; and in D, 10 µm, also applies to C.) Statistical analysis indicates that there was a significant decrease in spine density in the molecular layer of dentate gyrus of UBQLN2 transgenic mice compared with control mice (E, *P < 0.01). (F and G) Neuronal cell count in the granule cell layer of the dentate gyrus after cresyl violet and Luxol fast blue staining did not reveal any differences between transgenic and control mice (n = 3–4; P = 0.75). (Scale bar: 500 μm in F.) Extracellular recordings were performed from acute hippocampal slices in 3-mo-old transgenic mice and age-matched controls. (H) Average time course of the field potential (normalized to the pretrain response) recorded in CA1 stratum radiatum in response to stimulation (arrow) of the Schaffer collateral pathway. Inset traces: responses from individual slices from control (black traces) and transgenic (red traces) animals recorded before (thick traces) and 40 min after (thin traces) tetanic stimulation (four times 1-s long 100-Hz trains, 30-s intervals). (I) Summary of the type of synaptic plasticity observed 40 min after the tetanic stimulation in 8 control (black bars) and 10 transgenic (red bars) slices, measured from the excitatory postsynaptic potential (EPSP) slopes. The majority (75%) of the recordings from control mice produced LTP; this was not the case in transgenic animals, in which the same protocol induced LTP in only 13% of the cases. In slices from transgenic animals, the polarity of the synaptic modulation was actually reversed and led to LTD in the large majority (70%) of the recordings.

Fig. 6.

Proteasome subunits in protein aggregates of the UBQLN2P497H transgenic mice. (A–D) Immunohistochemistry was performed on hippocampal sections using antibodies to 26S proteasome subunits. Representative images show the protein aggregates containing subunits of 19S regulatory particle (ADRM1) and 20S enzymatic core particle (PSMA4, PSMB7, and PSMB3) in transgenic mice, but not in nontransgenic controls (Inset). (E–G) Colocalization of proteasome subunits, ubiquilin2 and ubiquitinated protein substrate in protein aggregates of the UBQLN2P497H/UbG76V–GFP double transgenic mice. Confocal microscopy was performed on the hippocampal sections of the UBQLN2P497H/UbG76V–GFP double transgenic mice using antibodies to ubiquilin2, proteasome subunits (PSMD4), and GFP as indicated. Protein aggregates containing proteasome subunits, ubiquilin2 and GFP are shown in the dentate gyrus (E–G) and CA3 (H–J). The majority of the protein aggregates showed weak to moderate (arrowhead) GFP immunoreactivity; some aggregates showed strong (arrow) GFP immunoreactivity (H–J).