Anterograde transsynaptic spread of pathological TDP-43 along pyramidal tract after intra-primary motor cortex injection of TDP-43 PFFs
We first examined the structure and morphology of TDP-43 PFFs using a transmission electron microscope (TEM), fluorescence spectroscopy with thioflavin T (ThT), and proteinase K (PK) resistance analysis. TEM analysis showed fibrillar protein with varying sizes, ranging from 120 to 800 nm length before sonication (Fig. 1a, c), and 40 to 480 nm after sonication (Fig. 1b, d). ThT fluorescence intensity was significantly increased after 5 min of phophate buffer incubation (Fig. 1e). Regarding TDP-43 PFFs seeding assay, the addition of 10% amounts of TDP-43 PFFs successfully triggered the fibrillation of soluble TDP-43 monomers. Without any agitation, TDP-43 monomers exhibited no increase in the intensity of ThT fluorescence and remained soluble for at least 3 days with no fibrillar aggregation. In contrast, even in the absence of agitation, the addition of 10% amounts of TDP-43 PFFs was sufficient to trigger the fibrillation of soluble TDP-43 monomers, which was confirmed by the increase in the ThT fluorescence intensity (Fig. 1f). PK resistance of TDP-43 PFFs was analyzed by the dot-blot test. The urea-fraction of TDP-43 solution dropped to significantly lower levels after agitation for 7 days (Fig. 1g). Furthermore, after a seeding reaction, a higher level of insoluble TDP-43 was detected in the pellet fraction, but less TDP-43 remained in the soluble fraction (Fig. 1h). These findings indicated the seeded fibrillation of TDP-43 PFFs in vitro.
To test whether the spreading of pathological TDP-43 via pyramidal tract can induce ALS-like phenotypes, we first generated a Thy1-e (IRES-TARDBP) 1 mouse line, specifically expressing hTDP-43 in nervous system, and evaluated the pathological phenotype of this mouse model on the behavior, molecular, and cellular levels. We detected hTDP-43 in M1, red nucleus (RN), inferior olive nucleus (ION), and cervical spinal cord anterior horn (Cs) of Thy1-e (IRES-TARDBP) 1 mice, and hTDP-43 was almost exclusively localized in the nucleus of neurons (Additional file 1: Fig. S1a-l). The expression level of hTDP-43 in Thy1-e (IRES-TARDBP) 1 mice were around 1.5-fold compared to the endogenous mouse TDP-43 (mTDP-43) in the cortex, and more than 2-fold in the spinal cord (Additional file 1: Fig. S1m-o). Moreover, pTDP-43-ir pathology was undetectable in PBS-injected Thy1-e (IRES-TARDBP) 1 mice at 8 mpi (Additional file 1: Fig. S2). Even at 20 mpi, no differences were found in rotarod test, hanging wire test, footprint test, Morris water maze test, weight, food and water consumption between Thy1-e (IRES-TARDBP) 1 mice and C57BL/6J mice (Additional file 1: Fig. S3).
To determine whether inoculation of TDP-43 PFFs in M1 region could induce transmission of TDP-43 pathology along pyramidal tract, we injected TDP-43 PFFs into two different parts of left M1 [45]: M1-C, representing forelimb and hindlimb movements; and M1-L, representing tongue and jaw movements in Thy1-e (IRES-TARDBP) 1 mice (Additional file 1: Fig. S4). The mice were sacrificed at 1, 3, 6, 8, and 12 mpi. The brain tissues were subjected to immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) to detect pathological TDP-43, total TDP-43 level and neuron loss. pTDP-43-immunoreactive (pTDP-43-ir) pathology was detected using an anti-pTDP-43 (phosphorylated at Ser409/Ser410) antibody at various time points. As early as 1 mpi, pTDP-43-ir pathology mainly distributed in the ipsilateral cortex around the injection regions in TDP-43 PFFs M1-C and M1-L mice, while pTDP-43-ir staining was not observed in the contralateral side. At 3 mpi, sparse cytoplasmic pTDP-43-ir staining was detected in the ipsilateral M1, secondary motor cortex (M2), primary somatosensory cortex (S1), field CA1 of hippocampus (CA1), internal capsule (IC), medullary reticular formation (MdRt), bilateral RN, hypoglossal nucleus (12N), and decussatio pyramidum (py) in TDP-43 PFFs M1-C mice. Simultaneously, pTDP-43-ir pathology was detected in the ipsilateral M1, IC, MdRt and bilateral 12N in TDP-43 PFFs M1-L mice. Over time, pTDP-43 pathology became much more extensive and possibly saturated. At 8 mpi, pTDP-43-ir staining was most abundant in bilateral M1, M2, S1, secondary somatosensory cortex (S2), cingulum (cg), CA1, field CA2 of hippocampus (CA2), field CA3 of hippocampus (CA3), pyramidal cell layer of the hippocampus, molecular layer of the dentate gyrus (Mol), IC, lateral globus pallidus (LGP), subthalamic nucleus (STh), reticular thalamic nucleus (Rt), stria terminalis (st), dorsomedial periaqueductal gray, interstitial nucleus of Cajal, substantia nigra (SN), medial lemniscus, cerebral peduncle, secondary visual cortex, temporal association cortex, MdRt, ION, paramedian reticular nucleus, RN, 12N, py, Cs, lumber spinal cord anterior horn (Ls) and ipsilateral agranular insular cortex, granular insular cortex, dysgranular insular cortex, piriform cortex, ectorhinal cortex, auditory cortex, perirhinal cortex, oriens layer of the hippocampus, dentate gyrus, zona incerta, optic tract, and lateral entorhinal cortex in TDP-43 PFFs M1-C mice (Fig. 2a–t). In addition, more severe pTDP-43 pathology was detected in ipsilateral brain regions to the injection site in M1, M2, S1, S2, cg, CA1, CA2, CA3, Mol, IC, LGP, Rt, st, and STh. However, in the contralateral regions, such as Cs and Ls, pTDP-43-ir pathological deposition became more severe than that in ipsilateral regions. In TDP-43 PFFs M1-L mice, pTDP-43 pathology became more abundant in bilateral 12N (Additional file 1: Fig. S5a-c). To further illustrate the spreading of TDP-43 pathology over time in TDP-43 PFFs M1-C mice, we produced a heat map to visualize the spatial distribution of pTDP-43 at different time points, as shown in Fig. 3. Additionally, the pTDP-43-ir staining was not detected in the brain of TDP-43 PFFs M1-injected C57BL/6J mice even at 20 mpi (Additional file 1: Fig. S6).
To investigate the deposition of pathological TDP-43 in different types of cells in the brain and spinal cord, we performed a series of double IF staining with pTDP-43 antibodies and specific cellular marker antibodies in different brain regions of TDP-43 PFFs M1-C mice at 4 mpi. We used microtubule-associated protein-2 (MAP-2) for neurons, glial fibrillary acidic protein (GFAP) for astrocytes, ionized calcium binding adapter molecule 1 (Iba-1) for microglia, myelin basic protein (MBP) for oligodendrocytes, and ubiquitin (Ub) for aggregates. Double IF analysis results revealed the co-localizations of pTDP-43 pathology with MAP-2 in M1 (Fig. 4a3) and parvicellular reticular nucleus (Fig. 4b3), ubiquitin in cervical spinal cord (Fig. 4c3), GFAP in LGP (Fig. 4d3), Iba-1 in medullary reticular nucleus and dorsal part (MdD) (Fig. 4e3). Neuronal cytoplasm contained the most abundant pTDP-43-positive aggregates (Fig. 4a3, b3), while few co-localizations of pTDP-43 with GFAP (Fig. 4d3) or Iba-1 (Fig. 4e3) were observed. Strong anti-ubiquitin signals were observed in neuron cytoplasm co-located with pTDP-43 (Fig. 4c3) in the Cs. Myelinolysis in the bilateral dorsal corticospinal tract was revealed by co-staining of MBP and NF (Fig. 4f1–g3). Obviously, the myelinolysis in the contralateral site was more pronounced than that in the ipsilateral site of TDP-43 PFFs injection. We semi-quantitatively detected the level of insoluble pTDP-43 over time in the cortex, RN, py, and cervical spinal cord of TDP-43 PFFs M1-C mice by WB. More insoluble pTDP-43 were detected in the extracts of TDP-43 PFFs M1-C mice compared with age-matched PBS M1-C mice at 4 mpi (Fig. 4h–o). In addition, we semi-quantitatively detected the insoluble C-terminal fragments of TDP-43 (CTFs) with time after injection in the cortex and Cs of TDP-43 PFFs M1-C and PBS M1-C mice. The WB analysis revealed increased levels of CTFs in both the cortex and Cs of TDP-43 PFFs M1-C mice at 4 and 8 mpi (Additional file 1: Fig. S7).
To detect whether TDP-43 PFFs M1-C mice developed age-dependent neuron loss, we performed stereology counts of Nissl staining in M1, ION, py and Cs in TDP-43 PFFs M1-C and PBS M1-C mice at 3 and 6 mpi, respectively. Furthermore, immunohistochemical staining for MAP-2 positive neurons in the M1, ION, py and calbindin (Cab) positive neurons in the Cs in TDP-43 PFFs M1-C mice at 3 and 6 mpi were also used to analyze the neuron loss. The findings indicated TDP-43 PFFs inoculation resulted in a increasingly significant decrease of neurons in TDP-43 PFFs M1-C mice as compared to PBS M1-C mice (Additional file 1: Fig. S8Aa-l and Fig. S8Ba-p). Moreover, we measured the brain wet weight of TDP-43 PFFs M1-C mice and PBS M1-C mice at 1, 3, 6, 8 mpi as reported previously [17]. The results showed that the TDP-43 PFFs M1-C mice presented obvious brain atrophy with age compared to the PBS M1-C mice (Additional file 1: Fig. S8C).
Motor deficits in TDP-43 PFFs M1-C mice
In addition to the neuropathological evidence of TDP-43 PFFs-induced damage, we also investigated whether the TDP-43 PFFs-injected mice exhibited motor dysfunction. The lifespan among TDP-43 PFFs M1-C mice, PBS M1-C mice, Thy1-e (IRES-TARDBP) 1 mice and C57BL/6J mice showed no significant difference (Additional file 1: Fig. S3r). In addition, motor functions were evaluated by a series of behavioral tests, including rotarod test, hanging wire test, and footprint test once a month from 1 to 12 mpi in TDP-43 PFFs M1-C and PBS M1-C mice. The rotarod test revealed reduced movements in TDP-43 PFFs M1-C mice from 2 mpi compared with PBS M1-C mice, while the PBS M1-C mice showed no movement reduction compared with C57BL/6J mice even at 12 mpi (Fig. 5a). As shown in Fig. 5b, the hanging wire test revealed a progressive loss of muscle strength, motor coordination, and balance in TDP-43 PFFs M1-C mice compared with PBS M1-C mice at 5 mpi. For the footprint test, TDP-43 PFFs M1-C mice exhibited shorter stride length, wider base width, and lower speed than age-matched PBS M1-C mice as early as 6 mpi (Fig. 5c–h), which demonstrated gait disturbances. Moreover, we found that the hind-climb muscles of TDP-43 PFFs M1-C mice were too weak to lift the pelvis off ground and lead dragging of hind-climb during the later period of footprint test at 6 mpi. What’s more, we performed biopsy on both sides of biceps brachialis muscles in TDP-43 PFFs M1-C mice at 6 mpi. Hematoxylin and eosin (H&E) staining showed abnormal changes including grouped atrophy, round muscle fibers, and muscle fibers with central nuclei in the right biceps brachialis muscle (Fig. 5i), while no obvious morphological changes were found in the contralateral biceps brachialis muscle. The age-matched PBS M1-C mice were used as a negative control, as shown in Fig. 5j. Analysis of size distribution of the fiber cross-sectional areas in the right biceps brachialis muscles showed a decreased proportion of myofibers with relatively larger diameter (900-1200 µm2), and an increased proportion of myofibers with relatively smaller diameter (0-600 µm2) in TDP-43 PFFs M1-C mice compared with PBS M1-C mice (Fig. 5k).
Learning and memory deficits in TDP-43 PFFs M1-C mice
In order to assess learning and memory, mice were tested in the Morris water maze. The escape latency in TDP-43 M1-C group was significantly longer than that in PBS M1-C group (Fig. 5l). Similar results were obtained when analyzing the path length to reach the platform (Fig. 5m). In addition, the swimming speed did not differ significantly among all groups (Fig. 5n). In the probe trial, spatial reference memory was strongly impaired in TDP-43 PFFs M1-C mice, and TDP-43 PFFs M1-C mice did not show a preference for the trained target quadrant in comparison with the PBS M1-C mice (Fig. 5o).
ALS-like neurophysiological phenotypes in TDP-43 PFFs M1-C mice
We next examined the electromyography (EMG) and motor evoked potentials (MEPs) of TDP-43 PFFs M1-C and PBS M1-C mice at 1, 3, 6, and 8 mpi. Spontaneous activity and motor unit action potentials (MUAPs) were used to quantify LMN dysfunction. MEPs were used to infer the mass activity of motor cortical neurons. Needle EMG was recorded from the bilateral biceps brachialis, tenth thoracic (T10) paraspinal, tibialis anterior, and gastrocnemius muscles. As early as 3 mpi, abnormal spontaneous activity including fibrillation potentials, fasciculation potentials, and positive sharp waves were detected in the right side of biceps brachialis, tibialis anterior, and gastrocnemius muscles of TDP-43 PFFs M1-C mice (Fig. 6a–d). Then, both sides of biceps brachialis, T10 paraspinals, tibialis anterior, and gastrocnemius muscles of TDP-43 PFFs M1-C mice were affected by abnormal spontaneous activity at 6 mpi (Fig. 6e). Nevertheless, in PBS M1-C mice, slight abnormal spontaneous activity emerged in bilateral biceps brachialis, T10 paraspinal, tibialis anterior, and gastrocnemius muscles at 8 mpi (Additional file 1: Fig. S9d). The frequency of abnormal spontaneous activity in TDP-43 PFFs M1-C mice was developed in a time-dependent manner, as shown in Fig. 6f. In addition, the motor unit measurement showed a significant increase in the mean amplitude and latency of MUAPs in bilateral biceps brachialis, T10 paraspinal, tibialis anterior, and gastrocnemius muscles at 6 mpi in TDP-43 PFFs M1-C mice, compared with age-matched PBS M1-C mice (Fig. 6g, h). Then, cortical MEP (cMEP) and spinal MEP (sMEP) were assessed to quantify upper motor neuron (UMN) impairment (Fig. 6i–l). At 6 mpi, L-cMEP amplitude decreased in TDP-43 PFFs M1-C mice compared with age-matched PBS M1-C mice (2.39 ± 0.34 mV versus 5.21 ± 0.24 mV, P < 0.001) at about 6 mpi (Fig. 6m). Moreover, central motor conduction time (CMCT) was significantly prolonged in the injection side in TDP-43 PFFs M1-C mice compared with age-matched PBS M1-C mice (4.49 ± 0.29 ms versus 3.05 ± 0.20 ms, P < 0.001), and the CMCT increased in a time-dependent manner (Fig. 6n). The statistical data of EMGs and MEP at all time points were shown in Additional file 1: Fig. S9. The above neurophysiological findings were served as preliminary evidence that TDP-43 PFFs injection caused ALS-like neurophysiological phenotypes.
Dysphagia symptom in TDP-43 PFFs M1-L mice
To further investigate whether the TDP-43 PFFs M1-L mice would suffer dysphagia after TDP-43 PFFs injection, we evaluated the swallowing function of TDP-43 PFFs M1-L and PBS M1-L mice. As early as 5 mpi, TDP-43 PFFs M1-L mice showed significant weight loss (Additional file 1: Fig. S5d). At 6 mpi, the consumption of food and water showed a significant downward trend in TDP-43 PFFs M1-L mice compared with that in age-matched PBS M1-L mice (Additional file 1: Fig. S5e, f). Furthermore, statistical data showed substantial declines in both lick and mastication rates compared with age-matched PBS M1-L mice (Additional file 1: Fig. S5g, h).