Analysis of endocannabinoid receptors and enzymes in the post-mortem motor cortex and spinal cord of amyotrophic lateral sclerosis patients

<p><i>Objective</i>: We have investigated the endocannabinoid system in the motor cortex of motor neuron disease (MND) patients. <i>Methods</i>: Post-mortem samples from MND patients and controls were used for immunostaining and/or Western blotting analysis of endocannabinoid elements. <i>Results</i>: We did not find any evidence of neuronal losses in the motor cortex of MND patients, but elevations in glial markers Iba-1 and GFAP were evident. We found no changes in FAAH and MAGL enzymes and in the CB₁ receptor, which correlated with the lack of cortical neuron death. By contrast, the Western blotting analysis of CB₂ receptors proved an increase in the motor cortex corroborated by immunostaining, correlating with the elevated gliosis in these patients. Double-labeling analyses revealed that this elevated CB₂ receptor immunostaining was located in GFAP-labelled astroglial cells. However, we also found CB₂ receptor labeling in cortical neurons confirmed with double immunofluorescence with the neuronal marker MAP-2. This was also found in the spinal cord, using double-labeling with the spinal motor neuron marker choline-acetyl transferase. This happened in both patients and controls, despite these neurons experienced an important degeneration in patients reflected in reduced Nissl staining, TDP-43 immunostaining and CB₁ receptor levels measured by Western blotting. <i>Conclusion</i>: We have confirmed that CB₂ receptors are elevated in the motor cortex of MND patients associated with the reactive gliosis. This phenomenon is previous to neuronal losses. We also found CB₂ receptors in cortical and spinal motor neurons. These observations support that targeting this receptor may serve for developing neuroprotective therapies in MNDs.</p

Histological and biochemical analysis of the FAAH enzyme in the striatum of SCA-3 transgenic and wild-type mice.

<p><u>Panels A and B</u>: qPCR analysis and Western blot quantification (in a representative blot) of the FAAH enzyme in the striatum of SCA-3 transgenic and wild-type mice at different stages of disease progression. <u>Panels C and D</u>: Immunostaining for the FAAH enzyme in the striatum of SCA-3 transgenic and wild-type mice at different stages of disease progression (bar = 50 μm). Arrows indicate stronger immunolabeling in surviving striatal neurons of SCA-3 transgenic mice. In all cases, the values are expressed as the mean ± SEM of more than 6 animals <i>per</i> group, assessing the data using a Student’s t-test: *p<0.05, **p<0.01 <i>versus</i> wild-type animals.</p

List of antibodies used in western blotting or immunohistochemical analyses (WB: Western blotting; IHC: Immunohistochemistry).

<p>List of antibodies used in western blotting or immunohistochemical analyses (WB: Western blotting; IHC: Immunohistochemistry).</p

TaqMan probes used.

<p>TaqMan probes used.</p

Biochemical analysis of endocannabinoid genes and proteins, and of related signaling lipids in the striatum of SCA-3 transgenic and wild-type mice.

<p><u>Panels A-D</u>: qPCR analysis of the CB₂ receptor and of the NAPE-PLD, DAGL and MAGL enzymes in the striatum of SCA-3 transgenic and wild-type mice at different stages of disease progression. <u>Panels E-I</u>: LC-MS analysis of the levels of anandamide (AEA) and its related congeners, palmitoylethanolamide (PEA) and oleylethanolamide (OEA), as well as 2-arachidonoylglycerol (2-AG) and its congener [2-oleylglycerol (2-OG), in the striatum of SCA-3 transgenic and wild-type mice at different stages of disease progression. In all cases, the values are expressed as the mean ± SEM of 5–6 subjects <i>per</i> group, assessing the data using a Student’s t-test.</p

Histological and biochemical analysis of the CB₁ receptor in the striatum of SCA-3 transgenic and wild-type mice.

<p><u>Panel A</u>: qPCR analysis of the CB₁ receptor in the striatum of SCA-3 transgenic and wild-type mice at different stages of disease progression. <u>Panels B and C</u>: Immunostaining for the CB₁ receptor in the striatum of SCA-3 transgenic and wild-type mice at different stages of disease progression (bar = 50 μm and 10 μm, respectively). The arrows indicate the presence of CB₁ receptor immunolabeling in corticostriatal terminals. In all cases, the values are expressed as the mean ± SEM of more than 6 mice <i>per</i> group, assessing the data using a Student’s t-test: *p<0.05, **p<0.01 <i>versus</i> wild-type animals.</p

MPTP treatment induces a similar lesion in striatum-substantia nigra of HO-1^−/− mice and their wild type littermates.

<p>Animals received one daily i.p. injection of saline or MPTP (30 mg/kg) for five consecutive days. Pictures show representative coronal sections, 40-µm thick, from VMB and STR stained with anti-TH antibody and counterstained with Nissl. <i>A</i>, Dopaminergic denervation in STR. <i>B</i>, Loss of dopaminergic neurons in VMB. Rectangles indicate SN. <i>C</i>, Stereological quantification of TH-immunoreactive neurons and Nissl positive neurons in SN. <i>D</i>, immunoblot of STR protein lysates. <i>Upper panel</i>, anti-TH antibody. <i>Lower panel</i>, anti-β-actin antibody showing similar protein load per lane. <i>E</i>, densitometric quantifications (arbitrary densitometry units) from representative immunoblots of D after normalization by β-actin densitometry units obtained from the same immunoblot.Results are representative of 5–10 animals per group. Values represent mean ± SD. Two-way ANOVA followed by Bonferroni's test was applied to determine the significance of biochemical differences among groups.</p

MPTP induces a similar deposition of ferric iron in Nrf2^−/− and HO-1^−/− mice.

<p>Iron precipitates were detected by DAB-enhanced Perls reaction followed by Nissl counterstaining. <i>A,</i> simplified scheme of a coronal section of midbrain showing the location of mouse dorsal and ventral substantia nigra on the left (SNc and SNr, respectively). The rectangle on the right side indicates the region shown in panels <i>D, E, F and G</i>). <i>B</i> and <i>C</i>, high magnification pictures of the boundary between SNc and SNr from wild type mice submitted to saline or MPTP treatments, respectively. Yellow arrowheads point some large Perls-negative cells with pale nuclei and dark nucleoli typical of dopaminergic neurons. Green arrowheads point small Perls-positive cells with picnotic nuclei typical of microglia. The red arrowhead points a Perls-positive microglial cell that has been drawn in the inset to show microgrial extensions. The large panels show representative fields of both SNc and SNr (location indicated in the rectangle of <i>A</i> panel), from saline-treated wild type mice (<i>D</i>), MPTP-treated wild type mice (<i>E</i>), MPTP-treated Nrf2^−/− mice (<i>F</i>), and MPTP-treated HO-1^−/− mice (<i>G</i>).</p

DA and DOPAC levels in STR of MPTP-treated Nrf2^−/− and HO-1^−/− and wild type littermate mice.

<p>Animals received one daily i.p. injection of saline or MPTP (30 mg/kg) for five consecutive days and striatal DA and DOPAC levels were determined by HPLC. <i>A and B,</i> the reduction in DA and DOPAC is more pronounced in MPTP-treated Nrf2^−/− mice than in MPTP-treated wild type littermates. <i>C and D,</i> the reduction in DA and DOPAC is similar between MPTP-treated HO-1^−/− mice and their wild type littermates. Values represent the mean ± SD from five samples. Two-way ANOVA followed by Bonferroni's test was applied to determine the significance of biochemical differences among groups. Asterisks denote significant differences between treatments with <i>P</i><0.05.</p

Sub-acute MPTP treatment induces a more profound lesion in STR and SN of Nrf2^−/− mice in comparison to their wild type littermates.

<p>Animals received one daily i.p. injection of saline or MPTP (30 mg/kg) for five consecutive days. Pictures show representative coronal sections, 40-µm thick, from VMB and STR stained with anti-TH antibody and counterstained with Nissl. <i>A</i>, Dopaminergic denervation in STR. <i>B</i>, Loss of dopaminergic neurons in VMB. Rectangles indicate SN. <i>C</i>, Stereological quantification of TH-immunoreactive neurons and Nissl positive neurons in SN. <i>D</i>, immunoblot of STR protein lysates. <i>Upper panel</i>, anti-TH antibody. <i>Lower panel</i>, anti-β-actin antibody showing similar protein load per lane. <i>E</i>, densitometric quantifications (arbitrary densitometry units) from representative immunoblots of D after normalization by β-actin densitometry units obtained from the same immunoblot. Results are representative of 5–10 animals per group. Values represent mean ± SD. Two-way ANOVA followed by Bonferroni's test was applied to determine the significance of biochemical differences among groups. Asterisks denote significant differences between treatments with <i>P</i><0.05.</p