Alzheimer disease and amyotrophic lateral sclerosis: an etiopathogenic connection

Abstract

The etiopathogenesis of neither the sporadic form of Alzheimer disease (AD) nor of amyotrophic lateral sclerosis (ALS) is well understood. The activity of protein phosphatase-2A (PP2A), which regulates the phosphorylation of tau and neurofilaments, is negatively regulated by the myeloid leukemia-associated protein SET, also known as inhibitor-2 of PP2A, I2(PP2A). In AD brain, PP2A activity is compromised, probably because I2(PP2A) is overexpressed and is selectively cleaved at asparagine 175 into an N-terminal fragment, I2NTF, and a C-terminal fragment, I2CTF, and both fragments inhibit PP2A. Here, we analyzed the spinal cords from ALS and control cases for I2(PP2A) cleavage and PP2A activity. As observed in AD brain, we found a selective increase in the cleavage of I2(PP2A) into I2NTF and I2CTF and inhibition of the activity and not the expression of PP2A in the spinal cords of ALS cases. To test the hypothesis that both AD and ALS could be triggered by I2CTF, a cleavage product of I2(PP2A), we transduced by intracerebroventricular injections newborn rats with adeno-associated virus serotype 1 (AAV1) containing human I2CTF. AAV1-I2CTF produced reference memory impairment and tau pathology, and intraneuronal accumulation of Aβ by 5-8 months, and motor deficit and hyperphosphorylation and proliferation of neurofilaments, tau and TDP-43 pathologies, degeneration and loss of motor neurons and axons in the spinal cord by 10-14 months in rats. These findings suggest a previously undiscovered etiopathogenic relationship between sporadic forms of AD and ALS that is linked to I2(PP2A) and the potential of I2(PP2A)-based therapeutics for these diseases.

Figures

Fig. 1. I2PP2A is cleaved into I2NTF and I2CTF and PP2A activity is compromised in spinal cords of sporadic ALS cases

A, C) Western blots developed with mAb to I2NTF and pAb to I2CTF show the cleavage of I2PP2A in spinal cords of ten cases of sporadic ALS and three age-matched controls. B, D) Quantitative analysis of the blots shows that the cleavage of I2PP2A into I2NTF and I2CTF (ratio of I2NTF or I2CTF to I2PP2A full-length) is markedly increased in sporadic ALS cases as compared with control group. E, F) PP2A activity determined by the phosphatase ELISA assay is markedly decreased in ALS cases compared with control group. G, H) Western blots developed with anti-PP2Ac mouse monoclonal antibody showed no significant change in the levels of the phosphatase in ALS cases when compared with control group I) β-actin as a loading control. J) PP2A activity inversely correlated to the level of I2CTF in the spinal cord. I2FL = I2PP2A full-length.

Fig. 2. AAV1-mediated gene expression of I2CTF inhibits PP2A activity without affecting the expression of the PP2A catalytic subunit PP2Ac in the brain and the spinal cord of rats

a: Study design and linear maps of the AAV vector plasmids (based on pTRUF12) employed. With the exception of the inverted terminal repeats (ITR), all viral genes had been removed and replaced with GFP or I2CTF and GFP. CMVe-CBA (cytomegalovirus enhancer-chicken beta actin promoter); IRES (internal ribosomal entry site) from poliovirus; b: Immunohistofluorescence with anti-GFP showed transduction of both brain and spinal cord; scale bars in cervical level, thoracic level and lumber level: 75 μm; scale bars in choroid plexus, hippocampus and cerebral cortex: 50 μm. c: Western blots developed with pAb to I2CTF showed a marked increase in the brain level of I2CTF in AAV1-I2CTF animals. d and e: Immunoprecipitation of PP2A from spinal cord (d) and brain (e) with rabbit polyclonal R123d anti-PP2Ac, followed by phosphatase activity assays showed inhibition of PP2A activity in I2CTF rats. b–e: Data from 14-month-old rats. *p<0.05; **p<0.01. Scale bar in b: cervical level, thoracic level and lumbar level, 75 μm; choroid plexus, hippocampus and cerebral cortex, 50 μm. I2FL = I2PP2A full-length.

Fig. 3. I2CTF expression induces spatial reference memory and neurological and motor impairments

a. Compared to GFP control (n=7), I2CTF (n=8) rats showed impairment in spatial reference memory at 5 (p=0.005) and at 8 (p<0.001) months of age in Morris Water Maze task and b. in long-term spatial reference memory examined one month later (at 9 months of age) by transfer task in the water maze (p=0.041). d=day; t=trial; asterisks refer to statistical difference between the two curves and not any single time point; *p<0.05; **p<0.01 and ***p<0.001. c: I2CTF rats (n=8) presented a higher neuroscore than GFP rats (n=7), reflecting a robust neurological impairment; d: I2CTF rats displayed severe muscle atrophy, inducing abnormal posture and hind limb clasping; e: weakness in prehensile strength; and f: disability in walking.

Fig. 4. I2CTF expression induces degeneration and loss of motor neurons, and degeneration and demyelination of axons, proliferation of neurofilaments, accumulation of tight fibrous bundles in the neuronal soma and axons at all levels of the spinal cord, and loss of Purkinje cells in the cerebellum

a–g: Nissl staining showed condensation (a–f), spongiosis (f) and loss (g) of motor neurons in the spinal cords of I2CTF rats. h (GFP rats), i (I2CTF rats): Toluidine blue-stained sections of the epon-embedded spinal cord showed a severe degeneration (i, asterisk), demyelination (i, arrowhead), and residues of degraded axons (i, double arrowheads) in I2CTF rats; j–m: Electron microscopy showed normal axons in GFP rats (j), and degenerated and demyelinated axons (k, arrowheads),inclusions of compact fibrous bundles in neuronal processes (l, arrowhead), 20–35 nm thick filaments in compact fibrous bundles (m, arrowhead) compared to ~10 nm neurofilaments (double arrowheads) and loss of morphology of fibers in the central portion of inclusions (m, asterisk). n–q: Toluidine blue-stained sections of the epon-embedded tissue showed an extensive cerebellar degeneration associated with cytoplasmic condensation and loss of Purkinje cells (o, q, arrowhead) in the I2CTF rats. n, p: GFP rats. r: quantitation of the numerical density of Purkinje cells from cresyl violet stained paraffin sections of cerebellar cortices from I2CTF and GFP rats. a–r: Data from 14-month-old rats. g, r: The data are presented as mean±SD. Scale bar: a–f: 50 μm; f: bar in inset: 10 μm; g, h: 15 nm; m, n: 50 nm; o, p: 25 nm; i, j: 2 μm; k: 500 nm; l: 100 nm.

Fig. 5. I2CTF expression induces hyperphosphorylation and accumulation of neurofilaments and tau, increase in the expression of ubiquitin, and aggregation and translocation of TD43 from the motor neuronal nucleus to the cytoplasm in I2CTF rats

a–f: Immunohistochemical staining with anti-non-phosphorylated (np) NF-H mAb (SMI33) showed a decrease in the density of npNF-H-positive motor neurons in the spinal cords of I2CTF rats; g–l: Immunohistochemical staining with anti-phospho (p) NF-H mAb (SMI34) showed an increase in phosphorylation and accumulation of neurofilaments in the axonal tracts of the spinal cords of I2CTF rats; m–r: Immunohistochemical staining with anti-pSer262/356 tau (mAb 12E8) showed abnormal hyperphosphorylation and aggregation of tau in the motor neuron cell cytoplasm reminiscent of stage 0 (r) and stage 1 (p) neurofibrillary tangles, and in dystrophic neurites resembling neuritic plaques (n); s–t: An increase in ubiquitin immunostaining was evident in the axonal tracts in the spinal cords of I2CTF rats; and u–v: Immunohistochemical staining showed condensation and translocation of TDP43 from the motor neuron nucleus to the cytoplasm in the spinal cords of I2CTF rats. a–v: Data from 14-month-old rats. Scale bar: 25 μm.

Fig. 6. Expression of I2CTF induces increase in abnormal hyperphosphorylation and accumulation of sarkosyl-insoluble tau in the brains of I2CTF rats

a: Immunohistofluorescent staining showed abnormal hyperphosphorylation of tau at pSer262/356 (12E8 site) in the CA3 and the subcortical area (SA) of I2CTF rats; b: Western blots and c: quantitative analysis showed a reduction in the level of total tau and an increase in the abnormal hyperphosphorylation of tau at multiple AD pathological sites and d: increase in sarkosyl-insoluble tau in the I2CTF rats’ hippocampus, cerebral cortex, subcortical area (SA) and cerebellum. a–e: Data from 14-month-old rats. Scale bar: a: 100 μm; inset: 25 μm.

Fig. 7. Expression of I2CTF causes neurodegeneration and loss of neuronal plasticity

a, b: Fluoro-Jade B staining showed an increase in neurodegeneration in I2CTF rats; c: Western blots and their quantitative analysis showed a decrease in the level of βIII tubulin and not in total tubulin in I2CTF rats; d, e: Immunohistofluorescent staining showed a decrease in the density of MAP2 in CA1 and CA3, synapsin 1 in CA3, and synaptophysin in CA3 of the hippocampi of I2CTF rats; and f: Western blots and their quantitative analysis showed a marked decrease in the levels of MAP2, synapsin 1, and synaptophysin in the I2CTF rats’ hippocampus, cerebral cortex, subcortical area (SA) and cerebellum. Data from 14-month-old rats. *p<0.05; **p<0.01. Scale bar: a: 300 μm; inset: 100 μm; d: 500 μm.

Fig. 8. I2CTF causes increase in the expression of Aβ1–40 and Aβ1–42 in the brain

Immunohistochemical staining showed an increase in the expression of Aβ as detected by a: anti-Aβ/APP (4G8), b: anti-Aβ1–40, and c: anti-Aβ1–42 in CA1, CA3, and dentate gyrus in the hippocampus, cerebral cortex, and in the subcortical area (SA) of I2CTF rats. Data from 14-month-old rats. Scale bar: a–c: 300 μm.