Is your doctor competent in providing this solution after you get Parkinsons post stroke? If not completely proven will your doctor ENSURE FINAL RESEARCH GETS DONE? NO? So, you DON'T have a functioning stroke doctor, do you?
Do you prefer your doctor incompetence NOT KNOWING? OR NOT DOING?
Parkinson’s Disease May Have Link to Stroke March 2017
The latest here:
A nanoparticle-based wireless deep brain stimulation system that reverses Parkinson’s disease
Wu et al., Sci. Adv. 11, eado4927 (2025) 15 January 2025
S c i e n c e A d v A n c e S | R e S e A R c h A R t i c l e
1 of 16
N E U R O S C I E N C E
Junguang Wu1,2,3, Xuejing Cui 1,3*, Lin Bao 19 release of DA (). Thus, we hypothesized that TRPV1 ion channels may serve as a modulatory target to activate DA neurons in the SN for PD therapy. Prior studies have demonstrated that the degeneration and death of DA neurons in PD are primarily attributed to the deposition of syn) fibrils, which form aggregates in the SN (25). Clearing these aggregates is a promising strategy for treating PD. The current approaches are mainly based on monoclo-nal antibodies with high affinity to syn fibrils (26); however, none of these drugs has successfully completed clinical trials. Therefore, there is a need for new treatments to restore DA neuron activity. Nanoparticles (NPs), such as graphene quantum dots and fullerenols, have been used to deconstruct aggregates via the charge interaction between NPs and syn fibrils (27-29), although this type of binding lacks specificity. In addition, an important factor contributing to the failure to clear-syn aggregates is the disruption of the autophagic system by the accumulation of α syn fibrils, leading to reduced levels of lysosomal enzymes or autophagic machinery (30-31). Restarting the intracellular autophagic process, such as the chaperone-mediated autophagy (CMA) pathway, is necessary for clearing pathologic syn. Therefore, an ideal therapeutic system for reducing the accumulation of neuronal-syn aggregates, which has been a great challenge, would simultaneously disaggregate α-synfibrils and initiate the autophagic process. Here, we designed a photothermal, wireless DBS nanosystem, termed Au@TRPV1@β-syn (ATB) NPs. It comprises three components: (i) gold nanoshells (AuNSs) for NIR light-to-(32–35), (ii) TRPV1 antibodies conjugated to AuNSs for specific targeting and activation of DA neurons, and (iii)β-synuclein (β-syn) peptides with a NIR-responsive linker for disaggregating α synfibrils through specific binding to the α-syn nonamyloid–β component hydrophobic domain. After entry into the SN by a single stereotaxic injection, ATB NPs anchored to DA neurons through the TRPV1 receptor. Upon pulsed NIR irradiation (808 nm), the ATB NPs, acting as nanoantennae, sensed and converted the light into heat, which effectively restored degenerated DA neurons by activating the heat-sensitive TRPV1 receptor, leading to elevated Ca2+ in-flux and action potentials. Concurrently, the NPs eliminated α- synaggregates and reduced pathological α-syn fibrils by releasing β- synpeptides and stimulating the CMA process. ATB NPs ultimately induced increased DA levels in the striatum and reversed locomotor behavior in α-syn preformed fibril (PFF)–induced PD mice. This “wireless” DBS therapeutic approach may open new avenues in the treatment of PD and other neurodegenerative diseases. RESULTS AND DISCUSSION ATB NPs activate and depolarize the TRPV1-positive cells First, we fabricated multifunctional ATB NPs that can achieve wire modules (Fig. 1A): (i) heat transfer module: AuNSs (with SiO2 core) due to surface plasmon resonance; (ii) targeting and activating module: conjugation with a specific TRPV1 antibody raised against the extracellular loop of TRPV1 (400 to 500 amino acids, containing the extracellular epitope 455 to 468 amino acids) for delivery into TRPV1- positive (TRPV1+) cell types in the SN; (iii) degrading module: linkage to a borate ester [NIR-responsive linker (36, 37)] containing β-syn peptides [36 to 45 amino acids, all D type, RTKS-GVYLVG (38); Fig. 1B], which will release the β- syn peptides into cells after NIR stimulation and disaggregate α-syn fibrils. We initially prepared and characterized the borate ester ligands formed between mercaptophenylboric acid (MPBA) and β- syn peptides (fig. S1, A to C). The Fourier transform infrared (FTIR) spectroscopy bands at 1417, 1365, and 1026 cm−1 indicate the presence of B─O bonds and borates (fig. S1A). The nuclear magnetic resonance(NMR) spectra in fig. S1B show a downfield shift of the protons on the benzene unit of MPBA after reacting with β-syn peptides to form boronic esters. This was due to the stronger electron-withdrawing property of the phenylboronate ester, leading to a decline of cloud density on the benzene unit. Consequently, the downfield shift of the protons occurs. The 11B NMR spectra in fig. S1C further demonstrate the transformation of boron from trihedral to tetrahedral, confirming the formation of a boronic ester. After TRPV1 and MPBA–β-syn peptide ligands were attached to the (1656 cm−1; amide bands appeared after AuNSs were modified with the TRPV1 antibody) and B─O bonds (1417, 1365, and 1026 cm−1 Fig. 1C). The diameter changes from ~166 nm for AuNSs to ~207 nm for ATB NPs indicate the conjugation of TRPV1 and β- syn to the NPs, as visualized by dynamic light scattering and transmission electron microscopy (TEM) (fig. S1, D and E). Moreover, a red shift in the maximum ultraviolet (UV) adsorption suggests the successfulof ATB NPs to respond to pulse NIR irradiation, we irradiated the NP suspensions with an 808-nm laser. ATB NPs showed excellent photothermal conversion performance under various laser powers and frequencies, even at low particle concentrations (fig. S1, G to I). Furthermore, the size of the NPs decreased (fig. S1J) and the infrared peak from the B─O bond reappeared after laser irradiation (fig.S1K), suggesting that β-syn peptides were released from ATB NPs due to the broken boronic ester caused after NIR irradiation. Overall, the fabricated ATB NPs enabled the generation of mild heat and release of β-syn peptides under the 808-nm NIR irradiation. We next investigated the ability of ATB NPs to target to the cell membrane of DA neurons. We first confirmed the presence of TRPV1 in isolated primary dopaminergic neurons (cultured for 7 days). The colocalization of TRPV1 with tyrosine hydroxylase (TH; DA neuron marker) indicated that mature DA neurons express endogenous markers, including class III β-tubulin (TuJ1), microtubule-associated protein 2 (MAP2), and vesicular monoamine transporter 2 (VMAT2; fig. S2A, middle and bottom). After incubation with primary DA neurons for 24 hours, ATB NPs did not induce cytotoxicity in DA neurons, even at the high concentration of 5 × 109 particles/ml (fig. S2B), suggesting an excellent biocompatibility of ATB NPs. We chose a concentration of 1 × 109 particles/ml for all subsequent cellular experiments. After incubation for 24 hours, ATB NPs were mainly located in the cell membrane of DA neurons (Fig. 1D, left). To verify that the highly expressed TRPV1 receptors on DA neurons mediate the anchoring of ATB NPs to the cell membrane, we transfected human embryonic kidney (HEK) 293T cells with a TRPV1 expression plasmid and incubated the cells with ATB NPs (1 × 10 9 /ml) for 24 hours. As expected, ATB NPs were present in the cytoplasm in TRPV1-negative (TRPV1−) cells, while TRPV1+ cells exhibited the most NPs around the cell membrane (Fig. 1D, right). We further visualized this TRPV1-mediated docking effect on the cell membranein α-syn PFF-induced degenerated DA neurons. After incubation with AuNSs or Au@TRPV1 NPs (AT NPs) for 24 hours, the AT NPs mainly localized to the cell membrane (fig. S2C). In contrast, most of the AuNSs, without TRPV1 antibody modification, were internalized by neurons. To further confirm the role of TRPV1 in ATB NPs binding to cell membranes, we blocked the TRPV1 channel with a specific TRPV1 antibody for 3 hours. Following a 24-hour incubation with ATB NPs, cells were observed using laser reflection technology of confocal laser scanning microscopy. In the absence of TRPV1 receptor, most ATB NPs were found in neuronal cells and axons(white arrows; fig. S2D). These data suggest that ATB NPs target the cell membranes of DA neurons through the TRPV1 receptors. We proceeded to explore whether ATB NPs and pulsed NIR stimuli can activate primary DA neurons in the presence of ATB NPs, using Ca2+ influx as an indicator of TRPV1 activity (Fig. 1E). Our treated DA neurons, as manifested by a continuous enhancement of the fluorescence intensity of Fluo-4 AM (Fig. 1F, right, and movies S1 to S3). The intracellular Ca2+ concentration in PFF-treated neurons remained unchanged in the absence of either the ATB NPs or external laser stimulation (Fig. 1F), indicating that ATB NPs only trigger Ca2+ influx under NIR laser stimulation. To confirm that the Ca2+ influx was mediated by TRPV1, we again transfected HEK-293T cells with a TRPV1 expression plasmid. Similarly, we found that ATB NPs and NIR laser stimulation promoted Ca2+ influx only in TRPV1+ cells but not in TRPV1− cells (Fig. 1G and movies S4 to S6). Thus, ATB NPs elicited Ca2+ influx under NIR laser stimulation through TRPV1. Consistent with the results from Ca2+ influx, we also observed that the ATB NPs induced inward currents in TRPV1+
S c i e n c e A d v A n c e S | R e S e A R c h A R t i c l e
1 of 16
N E U R O S C I E N C E
Junguang Wu1,2,3, Xuejing Cui 1,3*, Lin Bao 19 release of DA (). Thus, we hypothesized that TRPV1 ion channels may serve as a modulatory target to activate DA neurons in the SN for PD therapy. Prior studies have demonstrated that the degeneration and death of DA neurons in PD are primarily attributed to the deposition of syn) fibrils, which form aggregates in the SN (25). Clearing these aggregates is a promising strategy for treating PD. The current approaches are mainly based on monoclo-nal antibodies with high affinity to syn fibrils (26); however, none of these drugs has successfully completed clinical trials. Therefore, there is a need for new treatments to restore DA neuron activity. Nanoparticles (NPs), such as graphene quantum dots and fullerenols, have been used to deconstruct aggregates via the charge interaction between NPs and syn fibrils (27-29), although this type of binding lacks specificity. In addition, an important factor contributing to the failure to clear-syn aggregates is the disruption of the autophagic system by the accumulation of α syn fibrils, leading to reduced levels of lysosomal enzymes or autophagic machinery (30-31). Restarting the intracellular autophagic process, such as the chaperone-mediated autophagy (CMA) pathway, is necessary for clearing pathologic syn. Therefore, an ideal therapeutic system for reducing the accumulation of neuronal-syn aggregates, which has been a great challenge, would simultaneously disaggregate α-synfibrils and initiate the autophagic process. Here, we designed a photothermal, wireless DBS nanosystem, termed Au@TRPV1@β-syn (ATB) NPs. It comprises three components: (i) gold nanoshells (AuNSs) for NIR light-to-(32–35), (ii) TRPV1 antibodies conjugated to AuNSs for specific targeting and activation of DA neurons, and (iii)β-synuclein (β-syn) peptides with a NIR-responsive linker for disaggregating α synfibrils through specific binding to the α-syn nonamyloid–β component hydrophobic domain. After entry into the SN by a single stereotaxic injection, ATB NPs anchored to DA neurons through the TRPV1 receptor. Upon pulsed NIR irradiation (808 nm), the ATB NPs, acting as nanoantennae, sensed and converted the light into heat, which effectively restored degenerated DA neurons by activating the heat-sensitive TRPV1 receptor, leading to elevated Ca2+ in-flux and action potentials. Concurrently, the NPs eliminated α- synaggregates and reduced pathological α-syn fibrils by releasing β- synpeptides and stimulating the CMA process. ATB NPs ultimately induced increased DA levels in the striatum and reversed locomotor behavior in α-syn preformed fibril (PFF)–induced PD mice. This “wireless” DBS therapeutic approach may open new avenues in the treatment of PD and other neurodegenerative diseases. RESULTS AND DISCUSSION ATB NPs activate and depolarize the TRPV1-positive cells First, we fabricated multifunctional ATB NPs that can achieve wire modules (Fig. 1A): (i) heat transfer module: AuNSs (with SiO2 core) due to surface plasmon resonance; (ii) targeting and activating module: conjugation with a specific TRPV1 antibody raised against the extracellular loop of TRPV1 (400 to 500 amino acids, containing the extracellular epitope 455 to 468 amino acids) for delivery into TRPV1- positive (TRPV1+) cell types in the SN; (iii) degrading module: linkage to a borate ester [NIR-responsive linker (36, 37)] containing β-syn peptides [36 to 45 amino acids, all D type, RTKS-GVYLVG (38); Fig. 1B], which will release the β- syn peptides into cells after NIR stimulation and disaggregate α-syn fibrils. We initially prepared and characterized the borate ester ligands formed between mercaptophenylboric acid (MPBA) and β- syn peptides (fig. S1, A to C). The Fourier transform infrared (FTIR) spectroscopy bands at 1417, 1365, and 1026 cm−1 indicate the presence of B─O bonds and borates (fig. S1A). The nuclear magnetic resonance(NMR) spectra in fig. S1B show a downfield shift of the protons on the benzene unit of MPBA after reacting with β-syn peptides to form boronic esters. This was due to the stronger electron-withdrawing property of the phenylboronate ester, leading to a decline of cloud density on the benzene unit. Consequently, the downfield shift of the protons occurs. The 11B NMR spectra in fig. S1C further demonstrate the transformation of boron from trihedral to tetrahedral, confirming the formation of a boronic ester. After TRPV1 and MPBA–β-syn peptide ligands were attached to the (1656 cm−1; amide bands appeared after AuNSs were modified with the TRPV1 antibody) and B─O bonds (1417, 1365, and 1026 cm−1 Fig. 1C). The diameter changes from ~166 nm for AuNSs to ~207 nm for ATB NPs indicate the conjugation of TRPV1 and β- syn to the NPs, as visualized by dynamic light scattering and transmission electron microscopy (TEM) (fig. S1, D and E). Moreover, a red shift in the maximum ultraviolet (UV) adsorption suggests the successfulof ATB NPs to respond to pulse NIR irradiation, we irradiated the NP suspensions with an 808-nm laser. ATB NPs showed excellent photothermal conversion performance under various laser powers and frequencies, even at low particle concentrations (fig. S1, G to I). Furthermore, the size of the NPs decreased (fig. S1J) and the infrared peak from the B─O bond reappeared after laser irradiation (fig.S1K), suggesting that β-syn peptides were released from ATB NPs due to the broken boronic ester caused after NIR irradiation. Overall, the fabricated ATB NPs enabled the generation of mild heat and release of β-syn peptides under the 808-nm NIR irradiation. We next investigated the ability of ATB NPs to target to the cell membrane of DA neurons. We first confirmed the presence of TRPV1 in isolated primary dopaminergic neurons (cultured for 7 days). The colocalization of TRPV1 with tyrosine hydroxylase (TH; DA neuron marker) indicated that mature DA neurons express endogenous markers, including class III β-tubulin (TuJ1), microtubule-associated protein 2 (MAP2), and vesicular monoamine transporter 2 (VMAT2; fig. S2A, middle and bottom). After incubation with primary DA neurons for 24 hours, ATB NPs did not induce cytotoxicity in DA neurons, even at the high concentration of 5 × 109 particles/ml (fig. S2B), suggesting an excellent biocompatibility of ATB NPs. We chose a concentration of 1 × 109 particles/ml for all subsequent cellular experiments. After incubation for 24 hours, ATB NPs were mainly located in the cell membrane of DA neurons (Fig. 1D, left). To verify that the highly expressed TRPV1 receptors on DA neurons mediate the anchoring of ATB NPs to the cell membrane, we transfected human embryonic kidney (HEK) 293T cells with a TRPV1 expression plasmid and incubated the cells with ATB NPs (1 × 10 9 /ml) for 24 hours. As expected, ATB NPs were present in the cytoplasm in TRPV1-negative (TRPV1−) cells, while TRPV1+ cells exhibited the most NPs around the cell membrane (Fig. 1D, right). We further visualized this TRPV1-mediated docking effect on the cell membranein α-syn PFF-induced degenerated DA neurons. After incubation with AuNSs or Au@TRPV1 NPs (AT NPs) for 24 hours, the AT NPs mainly localized to the cell membrane (fig. S2C). In contrast, most of the AuNSs, without TRPV1 antibody modification, were internalized by neurons. To further confirm the role of TRPV1 in ATB NPs binding to cell membranes, we blocked the TRPV1 channel with a specific TRPV1 antibody for 3 hours. Following a 24-hour incubation with ATB NPs, cells were observed using laser reflection technology of confocal laser scanning microscopy. In the absence of TRPV1 receptor, most ATB NPs were found in neuronal cells and axons(white arrows; fig. S2D). These data suggest that ATB NPs target the cell membranes of DA neurons through the TRPV1 receptors. We proceeded to explore whether ATB NPs and pulsed NIR stimuli can activate primary DA neurons in the presence of ATB NPs, using Ca2+ influx as an indicator of TRPV1 activity (Fig. 1E). Our treated DA neurons, as manifested by a continuous enhancement of the fluorescence intensity of Fluo-4 AM (Fig. 1F, right, and movies S1 to S3). The intracellular Ca2+ concentration in PFF-treated neurons remained unchanged in the absence of either the ATB NPs or external laser stimulation (Fig. 1F), indicating that ATB NPs only trigger Ca2+ influx under NIR laser stimulation. To confirm that the Ca2+ influx was mediated by TRPV1, we again transfected HEK-293T cells with a TRPV1 expression plasmid. Similarly, we found that ATB NPs and NIR laser stimulation promoted Ca2+ influx only in TRPV1+ cells but not in TRPV1− cells (Fig. 1G and movies S4 to S6). Thus, ATB NPs elicited Ca2+ influx under NIR laser stimulation through TRPV1. Consistent with the results from Ca2+ influx, we also observed that the ATB NPs induced inward currents in TRPV1+
Images and more at link.
No comments:
Post a Comment